Erg monoclonal antibodies

ABSTRACT

Monoclonal antibodies, or antigen-binding fragments thereof, that bind to ERG, and more specifically, to an epitope formed by amino acids 42-66 of ERG3 are disclosed. The monoclonal antibodies can be non-human antibodies (e.g., rabbit or mouse) or humanized monoclonal antibodies having the CDR regions derived from those non-human antibodies. In other embodiments, the monoclonal antibodies are chimeric, having the light and heavy chain variable regions of a non-human ERG antibody. Methods of using the antibodies to detect ERG, or fusion proteins comprising all or part of an ERG polypeptide, such as an ERG polypeptide encoded by a TMPRSS2/ERG, SLC45A3/ERG, or NDRG1/ERG fusion transcript, are also provided, including methods of detecting ERG or ERG fusion events in a clinical setting. The antibodies can also be used to inhibit the activity of ERG or fusion proteins comprising all or part of an ERG polypeptide, such as an ERG polypeptide encoded by a TMPRSS2/ERG, SLC45A3/ERG, or NDRG1/ERG fusion transcript and to treat malignancies associated with overexpression of ERG or an ERG fusion event, such as prostate cancer, Ewing&#39;s sarcoma, acute myeloid leukemia, acute T-lymphoblastic leukemia, endothelial cancer, and colon cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/287,599 filed 27 May 2014, which is a continuation of U.S. patentapplication Ser. No. 13/266,908 filed 28 Oct. 2011 (now U.S. Pat. No.8,765,916), which is the U.S. National Phase application ofInternational Application PCT/US2010/032714, filed 28 Apr. 2010, whichclaims the benefit of, and relies on the filing date of, U.S.provisional patent application No. 61/173,834, filed 29 Apr. 2009, theentire disclosure of which is incorporated herein by reference.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under contract numberMDA905-04-R-0002 awarded by the Uniformed Services University and grantnumber CA162383 awarded by the National Institutes of Health and grantnumber W81XWH-08-1-0532 awarded by the U.S. Army Medical Research andMaterial Command. The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on 2 Feb. 2019, isnamed HMJ-109-US-02_Sequence_Listing.txt and is 8 Kilobytes in size.

BACKGROUND

The ETS-related gene (ERG) proto-oncogene is overexpressed in 60-70% ofprostate tumors as a result of recurring gene fusions involving TMPRSS2and the ETS family of genes (Petrovics et al., 2005; Tomlins et al.,2005; reviewed in Kumar-Sinha et al., 2008). Emerging studies on humanprostate cancer specimens and various experimental models underscore thecausative oncogenic function of ERG in prostate cancer (Klezovitch etal., 2008; Tomlins et al., 2008; Sun et al., 2008; Wang et al., 2008).Numerous reports highlighted both diagnostic and prognostic features ofthe genomic activation of ERG revealing that about half the prostatetumors harbor the most common gene fusion that takes place between theandrogen receptor-regulated TMPRSS2 gene promoter and ERG protein codingsequence (reviewed in Kumar-Sinha et al., 2008). Fusion between theTMPRSS2 gene promoter and ERG results in the overexpression ofN-terminally truncated or full-length forms of ERG (Klezovitch et al.,2008; Sun et al., 2008). Fusion events between erg and other androgeninducible promoter sequences, such as SLC45A3 (Han et al., 2008) andNDRG1 (Pflueger et al., 2009), have also been identified in prostatecancer.

Poor disease outcome for patients with tumors harboring duplications ofTMPRSS2/ERG fusions or chromosomal losses (Edel) associated with thefusion event has been highlighted (Attard et al., 2008; FitzGerald etal., 2008; Mehra et al., 2008) Current diagnosis of prostate cancer isbased on a variety of histological features, including architecturalgrowth pattern, loss of basal cells, nuclear atypia, amphophiliccytoplasm, intraluminal blue mucin, pink amorphous secretion, andmitotic figures (Egevad, 2008). If some of these features are notapparent, it can be difficult for pathologists to diagnose prostatecancer, especially in the case of prostate needle biopsies with verylimited tumor content (Mostofi et al., 1992; Mostofi et al., 1993).Molecular markers are now being used to aid diagnosis. For example,prostate cancer diagnosis can include basal cell staining of benignglands for specific cytokeratin or p63 (CK903, p63) and tumor cellassociated alpha-methyl acyl-CoA-Racemase (AMACR or P504S) (Luo et al.,2002; Rubin et al., 2002). However, these molecular markers have markedlimitations in routine diagnosis. Expression of AMACR is found in avariety of other non-malignant lesions, including up to 21% of benignprostatic glands, 58% of nephrogenic adenomas and approximately 18% ofcases of atypical adenomatous hyperplasia (Beach et al., 2002; Gupta etal., 2004; Jiang et al., 2001; Yang et al., 2002) New data evaluatingERG over expression and TMPRSS2/ERG genomic rearrangement are providinghighly promising new strategies in prostate cancer diagnosis andprognosis (Furusato et al., 2008; Saramaki et al., 2008).

EWS-ERG fusions have been described in a small subset of Ewing'ssarcoma, whereas ERG overexpression without fusion was highlighted inacute myeloid leukemia and acute T-lymphoblastic leukemia (Marcucci etal., 2005; Baldus et al., 2006). ERG overexpression has also been linkedto megakaryoblastic leukemia (Rainis et el. 2005). Other studies suggestthat increased ERG expression plays a role in Alzheimer's Disease (AD)and AD-like neuropathy in Down Syndrome (Shim et al., 2003; Ng et al.2010).

The structure of the human ERG gene includes at least 17 exons spanningapproximately 300 kilobases of genomic sequence and generating at leastnine separate transcripts. (Owczarek et al., 2004). The ERGJ-ERG5isoforms encode five separate polypeptides that can bind the ETS siteand act as transcriptional activators. (Owczarek et al., 2004). Of thesefive isoforms, ERG3 is the longest, encoding a 479 amino acidpolypeptide (SEQ ID NO: 1; Accession No. NP_891548.1). The ERG6-ERG9isoforms represent alternative splice forms with a different 5′ exonfrom other ERG isoforms. While ERG7 and ERG8 have open reading frames,ERG6 encodes multiple stop codons, suggesting that this ERG transcriptdoes not code for a functional protein. (Owczarek et al., 2004). TheERG9 transcript does not contain a putative start codon or a consensuspolyadenylation signal suggesting that it might also be a non-codingtranscript. (Owczarek et al., 2004).

Although, the ERG proto-oncogene was initially characterized more thantwenty years ago (Rao et al., 1987a; Rao et al., 1987b; Reddy et al.,1987), currently there is no available antibody for detecting ERG inclinical specimens. The ERG protein belongs to a highly homologous groupof proteins, the ETS (E-twenty six specific, E26 transformationspecific) multi-gene family of transcription factors, which areconserved throughout the metazoans (Turner and Watson, 2008). ETSproteins contain a winged helix-turn-helix DNA binding domain and apointed (SAM) domain implicated in protein-protein interaction. The highdegree of homology between members of this family presents a significantobstacle for raising an antibody against a specific member of thisprotein family.

Although polyclonal ERG antibodies are commercially available, theseantibodies exhibit low affinity for endogenous levels of the ERG proteinand high levels of non-specific staining that limit their usefulness,for example, in immunohistochemistry testing. As such, the commerciallyavailable polyclonal ERG antibodies are not suitable for detecting ERGproteins in clinical settings (e.g., in a tissue biopsy).

SUMMARY

The present disclosure provides antibodies that bind to human ERG andcan be used, for example, in methods of detecting and treating cancersassociated with ERG fusion events and/or ERG overexpression, such asprostate cancer. The antibodies exhibit high affinity for human ERG withlittle to no non-specific staining and thus are suitable for detectingERG proteins in clinical settings, including tissue biopsies, blood andurine.

One embodiment is directed to a monoclonal antibody that binds to humanERG and, more specifically, to an epitope formed by the followingpolypeptide sequence: GQTSKMSPRVPQQDWLSQPPARVTI, which corresponds toamino acid residues 42-66 of human ERG3 (SEQ ID NO:1) and is referred tohereinafter as the “ERG 42-66 epitope.” In another embodiment, theantibody is a monoclonal antibody that binds to human ERG, wherein theantibody's binding to human ERG3 (SEQ ID NO: 1) is competitivelyinhibited by a polypeptide that corresponds to amino acid residues 42-66of human ERG3.

In one embodiment, the antibody is a monoclonal antibody produced by thehybridoma clone 9FY (“9FY antibody”). In another embodiment, theantibody is a monoclonal antibody that binds to the ERG 42-66 epitopeand competitively inhibits the binding of the 9FY antibody to humanERG3.

In other embodiments, the monoclonal antibody is humanized, chimerizedor fully human. In one aspect, the monoclonal antibody comprises thecomplementarity determining regions (CDRs) of the light and heavy chainvariable regions of a non-human ERG antibody that binds the ERG 42-66epitope, which are joined to the framework (FR) regions of the light andheavy chain variable regions of a human antibody, and optionally joinedto the light and heavy chain constant regions of a human antibody. Inone embodiment, the monoclonal antibody binds to the ERG 42-66 epitopeand comprises a light chain variable domain comprising the three CDRs inSEQ ID NO:2 and a heavy chain variable domain comprising the three CDRsin SEQ ID NO:3. This humanized antibody retains the ERG antigenspecificity of the parental antibody, but is less immunogenic in a humansubject.

In another aspect, the antibody is a chimeric monoclonal antibody. Thechimeric antibody contains the light and heavy chain variable regions ofa non-human ERG antibody that binds the ERG 42-66 epitope, which areoptionally joined to the light and heavy chain constant regions of ahuman antibody. This chimeric antibody retains the ERG antigenspecificity of the parental antibody, but is less immunogenic in a humansubject. In one embodiment, the monoclonal antibody binds to the ERG342-66 epitope and comprises a light chain variable domain comprising theamino acid sequence of SEQ ID NO:2 and a heavy chain variable domaincomprising the amino acid sequence of SEQ ID NO:3.

Also contemplated is a fully human ERG antibody that binds to the ERG42-66 epitope.

The monoclonal antibodies described herein that bind to the ERG 42-66epitope can be used in a variety of research and medical applications.For example, the disclosure provides compositions comprising any one ofthe monoclonal antibodies described herein that bind to the ERG 42-66epitope and their use for detecting or treating cancers or pathologicconditions associated with an ERG fusion event or ERG overexpression,including, for example, prostate cancer, Ewing's sarcoma, acute myeloidleukemia, megakaryoblastic leukemia, endothelial cancer, and acuteT-lymphoblastic leukemia. Monoclonal antibodies that bind to the ERG42-66 epitope can also be used to detect human ERG or a fusion proteincomprising all or part of a human ERG polypeptide, such as an ERGprotein encoded by a TMPRSS2/ERG fusion transcript, in a biologicalsample.

Another aspect is related to compositions comprising the ERG 42-66polypeptide. In one embodiment, the composition further comprises anadjuvant and/or a hapten, such as KLH, coupled to the ERG 42-66polypeptide. These compositions can be used, for example, in a method ofproducing antibodies. In one embodiment, the method comprisesadministering the composition to a non-human mammal, including but notlimited to a mouse or rabbit. The method may further comprise isolatingB cells from the non-human mammal, immortalizing the B cells to create acell line capable of producing a monoclonal antibody, and selecting themonoclonal antibody that binds to the ERG 42-66 epitope.

In a further aspect, the invention provides isolated polynucleotidesthat comprise nucleic acid sequences encoding the amino acid sequencesof one or more of the CDRs of the light and/or heavy chain variableregions of a monoclonal antibody described herein that binds to the ERG42-66 epitope, including, for example, the 9FY antibody. Similarly, theinvention provides isolated polynucleotides that comprise DNA sequencesencoding the amino acid sequence of the light and/or heavy chainvariable regions of a monoclonal antibody described herein that binds tothe ERG 42-66 epitope, including, for example, the 9FY antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate certain embodiments, and togetherwith the written description, serve to explain certain principles of theantibodies and methods disclosed herein.

FIG. 1A shows that the 9FY antibody detects the endogenous ERG proteinencoded by the TMPRSS2/ERG3 fusion transcript (50-52 kDa) in immunoblotsof TMPRSS2/ERG3 fusion harboring VCaP cells, expressed in response tosynthetic androgen hormone (R1881) treatment (lanes 4-6). 9FY antibodydoes not show immuno-reactivity to LNCaP cells that do not harbor aTMPRSS2/ERG fusion protein (lanes 1-3). On the bottom panels, hormonedose dependent expression of prostein (SLC45A3) and PSA (KLK3) areshown. FIG. 1B shows that ERG siRNA inhibits ERG expression in R1881treated VCaP cells.

FIG. 2A shows that the 9FY antibody recognizes the immunizingpolypeptide in an ELISA assay. Various concentrations of the immunizingpolypeptide and two control polypeptides were plated in wells for ELISAand were probed with 1:10,000 dilution of 9FY antibody. Y-axis indicatesabsorbance values at 450 nm. On the X-axis the log 10 concentration ofthe ERG immunizing polypeptide and control polypeptides is shown inng/ml. FIG. 2B shows that the immunizing polypeptide competes with ERG3protein for 9FY antibody binding. In sandwich ELISA assay, ERG3 proteinlysates were plated in multi-well format plates and were assayed withvarious concentrations of 9FY antibody in the presence (white bar) orabsence of the immunizing polypeptide (shaded bar). Fold dilution ismarked on the X-axis. Antibody titration values are marked by shadedbars.

FIG. 3 shows that the 9FY antibody recognizes and immunoprecipitates ERGprotein from genomic target sites as shown by in vivo chromatinimmunoprecipitation assay (ChIP). R1881 treated VCaP cells weretransfected by either ERG siRNA (si) to knock-down ERG or by controlNon-targeting (NT) siRNA. Cells were processed for ChIP assay and thechromatin was immunoprecipitated by using 9FY antibody. In control (NT)transfected cells ERG protein is recruited to HPGDH, C-MYC, KLK3 (PSA)and SLC45A3 (prostein) genomic target sequences. ERG knockdown stronglyreduces ERG recruitment to the genomic target sites. Input genomic DNAamplicons were used as controls for the ChIP assay.

FIGS. 4A-E show that the 9FY antibody specifically recognizes endogenousERG protein in an immunofluorescence (IF) assay. ERG is stained byprimary 9FY antibody followed by goat anti-mouse ALEXA FLUOR® 594(Invitrogen, Carlsbad, Calif.) secondary antibody (red). The androgeninducible PSA that is negatively controlled by ERG is stained by primaryrabbit polyclonal anti-PSA followed by goat anti-rabbit ALEXA FLUOR® 488(Invitrogen, Carlsbad, Calif.) secondary antibody (green). DNA isstained by DAPI (blue). Merged images in the right columns show nuclearlocalization of ERG. FIG. 4A shows that ERG knockdown with ERG siRNAeliminates ERG and nuclear 9FY antibody staining from R1881 treated VCaPcells. Cytoplasmic PSA expression is consistent with the hormoneinduction of VCaP cells. FIG. 4B shows that the immunizing polypeptide(Competing peptide) eliminates ERG staining with 9FY antibody. Incontrast, a control polypeptide (Non-competing peptide) does not affect9FY antibody staining. FIG. 4C shows that the 9FY antibody detects therobust activation of ERG expression in response to androgen hormone(R1881) in TMPRSS2/ERG fusion harboring VCaP cells but not in LNCaPcells that do not express endogenous ERG. FIG. 4D shows that 9FYantibody staining is reduced in VCaP cells in response to ERG knockdownwith ERG siRNA. PSA expression is increased in response to ERG knockdownthat is consistent with the negative regulatory role of ERG on theexpression of PSA. 9FY antibody detects ERG even in the 1:5,000 to1:20,000 fold dilution ranges of the 9FY antibody stock solution of 3.7mg/ml. FIG. 4E shows that performing an IF assay in the absence of 9FYantibody and in the presence of the secondary antibody yields nobackground staining.

FIGS. 5A-D show that the 9FY antibody specifically stains prostate tumorepithelial cells in formalin fixed paraffin embedded (FFPE) humanprostate radical prostatectomy specimens assayed by immunohistochemistry(IHC). FIG. 5A shows representative view fields of prostate sectionsstained with 9FY antibody. Superior distinction between prostate tumorepithelium and benign epithelium is evident in IHC. The lack of stainingof prostatic stroma is also evident. Vascular endothelia that expresswild-type ERG are stained by 9FY antibody and present an excellentinternal reference for the proper function of 9FY antibody. FIG. 5Bshows a high magnification view of an FFPE section and the clearstaining of prostate tumor epithelium by the 9FY antibody. FIG. 5C showsthe panoramic view of ERG staining by 9FY antibody on a section offormalin fixed paraffin embedded (FFPE) human prostate radicalprostatectomy specimen. FIG. 5D shows the corresponding hematoxylin andeosin staining of the section.

FIG. 6 shows that endogenously expressed ERG protein is detected by 9FYantibody in colon cancer (COLO 320), acute myeloid leukemia (KG1), andhuman acute T lymphoblast leukemia (MOLT4) derived cell lines by usingimmunoblot assay.

FIG. 7 shows that the 9FY antibody specifically detects exogenouslyoverexpressed ERG protein encoded by a TMPRSS2/ERG fusion transcript inimmunoblots, whereas commercially available polyclonal antibodies detectmultiple proteins in the same cell lysates. 20 gig of control HEK293cell lysates transfected with empty vectors were loaded in lanes 1, 4and 7; 4 μg and 20 gig of lysates from pIRES-TMPRSS2-ERG3 transfectedHEK293 cells were loaded in lanes 2, 5 and 8, and lanes 3, 6 and 9,respectively.

FIG. 8A shows that the polyclonal H95 (sc-28680) antibodies detectmultiple non-specific bands in LNCaP and VCaP cell lysates. LNCaP andVCaP cells were treated with (lanes 2, 3, 5, and 6) or without (lanes 1and 4) the synthetic androgen hormone (R1881). FIG. 8B shows thenon-specific binding of H95 antibodies to unknown proteins in VCaP cellstreated with ERG siRNA and/or Non-targeting (NT) RNA. In the absence ofERG siRNA, H95 antibodies do not appear to detect ERG expression.Furthermore, H95 antibodies do not permit one to observe the inhibitionof ERG expression by ERG siRNA.

FIGS. 9A and B shows that the commercially available C-20 polyclonalantibodies (sc-353) recognize not only nuclear ERG protein but alsonon-specific cytoplasmic proteins in an IF assay.

FIG. 10 shows that the commercially available H95 polyclonal antibodies(sc-28680) exhibit non-specific nuclear and cytoplasmic staining in bothVCaP and LNCaP cells in an IF assay.

FIG. 11 shows that the commercially available R20 polyclonal antibodies(sc-18136) exhibit non-specific nuclear and cytoplasmic staining in bothVCaP and LNCaP cells in an IF assay.

FIG. 12 shows the results of a competitive IF assay in VCaP cellsbetween the 9FY antibody (ERG-MAb) and the commercially available H95polyclonal antibodies (sc-28680).

FIG. 13 shows that the 9FY antibody detects ERG oncoprotein productsencoded by common splice variants of TMPRSS2-ERG fusions. Specifically,the 9FY antibody recognized ERG oncoprotein products encoded by:TMPRSS2-ERG fusion type-A cDNA (lanes 2 and 3), wild type ERG3 cDNA(lane 4) and TMPRSS2-ERG8 fusion type-A cDNA (lane 5).

FIGS. 14A-D show the results of a comparative immunohistochemistryanalysis between the 9FY antibody and the commercially available C20 (sc353) antibody (Santa Cruz Biotechnology, Inc, Santa Cruz, Calif.) inprostate tissues obtained from wild type and ERG transgenic mice. FIGS.14A and 14C show 9FY antibody staining in wild type and ERG transgenicprostate tissue, respectively. Solid arrows mark prostate glands anddashed arrows indicate endothelial cells. FIGS. 14B and 14D show thestaining patterns of the commercially available C20 (sc 353) antibody inwild type and ERG transgenic prostate tissue, respectively.

FIG. 15 shows a schematic representation of the expression of ERGoncoprotein (IHC) and TMPRSS2-ERG fusion mRNA in prostate tumors of 35patients treated with radical prostatectomy by using a branched-chainDNA assay. Consecutive tissue slides from whole mounted FFPE prostatespecimens were used for the two assays in a blinded fashion. Backwardslash marked triangles represent positive ERG oncoprotein staining,while forward slash marked triangles represent the detection ofTMPRSS2-ERG fusion mRNA. Hollow triangles indicate specimens with no ERGoncoprotein or with non-detectable TMPRSS2-ERG fusion transcript.

DETAILED DESCRIPTION

Reference will now be made in detail to various exemplary embodiments,examples of which are illustrated in the accompanying drawings. It is tobe understood that the following detailed description is provided togive the reader a fuller understanding of certain embodiments, features,and details of aspects of the invention, and should not be interpretedas a limitation of the scope of the invention.

1. Definitions

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

The term “antibody” as used in this disclosure refers to animmunoglobulin or an antigen-binding fragment thereof. The term includesbut is not limited to polyclonal, monoclonal, monospecific,polyspecific, non-specific, humanized, human, single-chain, chimeric,synthetic, recombinant, hybrid, mutated, grafted, and in vitro generatedantibodies. The antibody can include a constant region, or a portionthereof, such as the kappa, lambda, alpha, gamma, delta, epsilon and muconstant region genes. For example, heavy chain constant regions of thevarious isotypes can be used, including: IgG₁, IgG₂, IgG₃, IgG₄, IgM,IgA₁, IgA₂, IgD, and IgE. By way of example, the light chain constantregion can be kappa or lambda.

The terms “antigen-binding domain” and “antigen-binding fragment” referto a part of an antibody molecule that comprises amino acids responsiblefor the specific binding between antibody and antigen. For certainantigens, the antigen-binding domain or antigen-binding fragment mayonly bind to a part of the antigen. The part of the antigen that isspecifically recognized and bound by the antibody is referred to as the“epitope” or “antigenic determinant.” Antigen-binding domains andantigen-binding fragments include Fab (Fragment antigen-inding); aF(ab′)₂ fragment, a bivalent fragment having two Fab fragments linked bya disulfide bridge at the hinge region; Fv fragment; a single chain Fvfragment (scFv) see e.g., Bird et al. (1988) Science 242:423-426; andHuston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); a Fdfragment having the two V_(H) and C_(H)1 domains; dAb (Ward et al.,(1989) Nature 341:544-546), and other antibody fragments that retainantigen-binding function. The Fab fragment has V_(H)-C_(H)1 andV_(L)-C_(L) domains covalently linked by a disulfide bond between theconstant regions. The F_(v) fragment is smaller and has V_(H) and V_(L)domains non-covalently linked. To overcome the tendency ofnon-covalently linked domains to dissociate, a scF_(v) can beconstructed. The scF_(v) contains a flexible polypeptide that links (1)the C-terminus of V_(H) to the N-terminus of V_(L), or (2) theC-terminus of V_(L) to the N-terminus of V_(H). A 15-mer (Gly₄Ser)₃peptide may be used as a linker, but other linkers are known in the art.These antibody fragments are obtained using conventional techniquesknown to those with skill in the art, and the fragments are evaluatedfor function in the same manner as are intact antibodies.

The term “effective amount” refers to a dosage or amount that issufficient to reduce the activity of ERG or a fusion protein comprisingall or part of a human ERG polypeptide, such as an ERG protein encodedby a TMPRSS2/ERG fusion transcript, to result in amelioration ofsymptoms in a patient or to achieve a desired biological outcome. Forexample, with prostate cancer the desired biological outcome may includea decrease in tumor size, a decrease in Gleason score, and/or increasedtumor differentiation.

The term “ERG protein encoded by a TMPRSS2/ERG fusion transcript” refersto a truncated ERG protein, such as ERG3, encoded by a transcriptgenerated by a fusion event between a TMPRSS2 gene promoter and a humanERG transcript.

The term “fusion protein” refers to a protein translated from atranscript generated from a fusion event between two nucleotidesequences. One of the nucleotide sequences may be a non-coding sequence(e.g., a TMPRSS2, SLC45A3, or NDRG1 sequence) while the other nucleotidesequence represents all or part of a coding a sequence (e.g., ERG).Alternatively the two nucleotide sequences may both be coding sequences.

The term “human antibody” includes antibodies having variable andconstant regions corresponding substantially to human germlineimmunoglobulin sequences known in the art, including, for example, thosedescribed by Kabat et al. (See Kabat, et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242), which is herebyincorporated by reference in its entirety. Human antibodies may includeamino acid residues not encoded by human germline immunoglobulinsequences (e.g., mutations introduced by random or site-specificmutagenesis in vitro or by somatic mutation in vivo), for example in theCDRs, and in particular, CDR3. The human antibody can have at least one,two, three, four, five, or more positions replaced with an amino acidresidue that is not encoded by the human germline immunoglobulinsequence.

The terms “treatment” or “treating” and the like refer to any treatmentof any disease or condition in a mammal, e.g. particularly a human or amouse, and includes inhibiting a disease, condition, or symptom of adisease or condition, e.g., arresting its development and/or delayingits onset or manifestation in the patient or relieving a disease,condition, or symptom of a disease or condition, e.g., causingregression of the condition or disease and/or its symptoms.

The terms “subject,” “host,” “patient,” and “individual” are usedinterchangeably herein to refer to any mammalian subject for whomdiagnosis or therapy is desired, particularly humans.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” means solvents, dispersion media, coatings,antibacterial agents and antifungal agents, isotonic agents, andabsorption delaying agents, and the like, that are compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art.

The term “isolated,” when used in the context of an antibody, refers toan antibody that is substantially free of its natural environment. Forinstance, an isolated protein is substantially free of cellular materialor other proteins from the cell or tissue source from which it wasderived. The term also refers to preparations where the isolated proteinis sufficiently pure for pharmaceutical compositions; or at least 70-80%(w/w) pure; or at least 80-90% (w/w) pure; or at least 90-95% pure; orat least 95%, 96%, 97%, 98%, 99%, or 100% (w/w) pure.

The term “9FY antibody” is a monoclonal antibody produced by thehybridoma clone named 9FY that has been maintained through successivecell culture passages in the laboratory at the Center for ProstateDisease Research in Rockville, Md. since Sep. 17, 2008. Aliquots of the9FY hybridoma clone have also been labeled, frozen, and stored at theCenter for Prostate Disease Research.

The term “ERG 42-66 epitope” refers to a conformational or linearepitope formed by amino acid residues 42-66 of SEQ ID NO: 1.

The term “ERG 42-66 polypeptide” refers to a polypeptide having no morethan 60 amino acid residues, wherein the polypeptide includes amino acidresidues 42-66 of SEQ ID NO: 1. In certain embodiments, the ERG 42-66polypeptide has no more than 50, 40, 30, or 25 amino acid residues.

The term “human ERG3” refers to a polypeptide having the amino acidsequence of ACCESSION NP_891548; VERSION NP_891548.1 GI:33667107:

(SEQ ID NO: 1) 1 mastikeals vvsedqslfe caygtphlak temtassssdygqtskmspr vpqqdwlsqp 61 parvtikmec npsqvngsrn spdecsvakg gkmvgspdtvgmnygsymee khmpppnmtt 121 nerrvivpad ptlwstdhvr qwlewavkey glpdvnillfqnidgkelck mtkddfqrlt 181 psynadills hlhylretpl phltsddvdk alqnsprlmharntggaafi fpntsvypea 241 tqrittrpdl pyepprrsaw tghghptpqs kaaqpspstvpktedqrpql dpyqilgpts 301 srlanpgsgq iqlwqfllel lsdssnssci twegtngefkmtdpdevarr wgerkskpnm 361 nydklsralr yyydknimtk vhgkryaykf dfhgiaqalqphppesslyk ypsdlpymgs 421 yhahpqkmnf vaphppalpv tsssffaapn pywnsptggiypntrlptsh mpshlgtyy

2. Anti-ERG Antibodies

As discussed above, ERG is a proto-oncogene that belongs to the ETSfamily of transcription factors. Because of high degree of homologyamong members of the ETS family of proteins, raising an antibody againsta specific member of the family remains a challenge. Furthermore, todate, polyclonal antibodies raised against ERG retain significantnon-specific binding, preventing such antibodies from being used in aclinical setting (e.g., IHC) to aid in the detection of cancerous cellsor tissue expressing ERG or fusion proteins comprising all or part of anERG polypeptide, such as an ERG polypeptide encoded by a TMPRSS2/ERG,SLC45A3/ERG, or NDRG1/ERG fusion transcript that have been identified inprostate cancers.

This disclosure provides antibodies that bind to human ERG. Morespecifically, the antibodies bind to the ERG 42-66 epitope. Amino acids42-66 of human ERG3 are encoded by a nucleotide sequence that maps toexon 8 of the ERG locus (Owczarek et al., 2004). Exon 8 is found inERG2, ERG3, ERG4, ERG5 (partial exon), ERG7, and ERG8 but not in ERG1(Owczarek et al., 2004). Thus, the antibodies disclosed herein do notcross react with ERG1 protein.

Antibodies, also known as immunoglobulins, are typically tetramericglycosylated proteins composed of two light (L) chains of approximately25 kDa each and two heavy (H) chains of approximately 50 kDa each. Twotypes of light chain, termed lambda and kappa, may be found inantibodies. Depending on the amino acid sequence of the constant domainof heavy chains, immunoglobulins can be assigned to five major classes:A, D, E, G, and M, and several of these may be further divided intosubclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂.Each light chain includes an N-terminal variable (V) domain (V_(L)) anda constant (C) domain (C_(L)). Each heavy chain includes an N-terminal Vdomain (VH), three or four C domains (CHs), and a hinge region. The CHdomain most proximal to VH is designated as CHI. The VH and VL domainsconsist of four regions of relatively conserved sequences calledframework regions (FR1, FR2, FR3, and FR4), which form a scaffold forthree regions of hypervariable sequences (complementarity determiningregions, CDRs). The CDRs contain most of the residues responsible forspecific interactions of the antibody with the antigen. CDRs arereferred to as CDR1, CDR2, and CDR3. Accordingly, CDR constituents onthe heavy chain are referred to as H1, H2, and H3, while CDRconstituents on the light chain are referred to as L1, L2, and L3.Identification and numbering of framework and CDR residues is asdescribed by Chothia et al., Structural determinants in the sequences ofimmunoglobulin variable domain, J Mol Biol 1998, 278:457-79, which ishereby incorporated by reference in its entirety.

CDR3 is typically the greatest source of molecular diversity within theantibody-binding site. H3, for example, can be as short as two aminoacid residues or greater than 26 amino acids. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known in the art. For a review of the antibody structure, seeAntibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, eds.Harlow et al., 1988. One of skill in the art will recognize that eachsubunit structure, e.g., a CH, VH, CL, VL, CDR, FR structure, comprisesactive fragments, e.g., the portion of the VH, VL, or CDR subunit thebinds to the antigen, i.e., the antigen-binding fragment, or, e.g., theportion of the CH subunit that binds to and/or activates, e.g., an Fcreceptor and/or complement. The CDRs typically refer to the Kabat CDRs,as described in Sequences of Proteins of Immunological Interest. USDepartment of Health and Human Services (1991), eds. Kabat et al.Another standard for characterizing the antigen binding site is to referto the hypervariable loops as described by Chothia. See. e.g., Chothia,D. et al. (1992) J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995)EMBO J. 14:4628-4638. Still another standard is the AbM definition usedby Oxford Molecular's AbM antibody modelling software. See, generally,e.g., Protein Sequence and Structure Analysis ofAntibody VariableDomains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. andKontermann, R., Springer-Verlag, Heidelberg). Embodiments described withrespect to Kabat CDRs can alternatively be implemented using similardescribed relationships with respect to Chothia hypervariable loops orto the AbM-defined loops.

In one embodiment, the antibody is a monoclonal antibody that binds tothe ERG 42-66 epitope and is produced by the hybridoma clone 9FY (“9FYantibody”). The light chain variable domain of the 9FY antibodycomprises the amino acid sequence of SEQ ID NO:2. The amino acidsequences of the CDR1, CDR2, and CDR3 of the light chain variable domainof the 9FY antibody correspond to SSVYY (SEQ ID NO:4), YTS, andLQFSTSPWT (SEQ ID NO:5), respectively. The heavy chain variable domainof the 9FY antibody comprises the amino acid sequence of SEQ ID NO:3.The amino acid sequences of the CDR1, CDR2, and CDR3 of the heavy chainvariable domain of the 9FY antibody correspond to GYTFTNYG (SEQ IDNO:6), IDTYTGEP (SEQ ID NO:7), and VRKRAYDYEIY (SEQ ID NO:8),respectively.

In another embodiment, the antibody is a monoclonal antibody that bindsto the ERG 42-66 epitope and competitively inhibits the binding of the9FY antibody to human ERG3. In yet another embodiment, the antibody is amonoclonal antibody that binds to the ERG 42-66 epitope andcompetitively inhibits the binding of an antibody, having a variablelight chain comprising the amino acid sequence of SEQ ID NO:2 and avariable heavy chain comprising the amino acid sequence of SEQ ID NO:3,to human ERG3. Whether an antibody competitively inhibits the binding ofan antibody to a protein, such as human ERG3, can be assessed usingroutine methods in the art, including, for example, the methodsdescribed in the examples of this application.

Antibodies, in which CDR sequences or heavy or light chain variabledomains differ only insubstantially from those of the 9FY antibody arealso contemplated. Typically, an amino acid is substituted by a relatedamino acid having similar charge, hydrophobic, or stereochemicalcharacteristics. Such substitutions would be within the ordinary skillsof an artisan. Unlike in CDRs, more substantial changes can be made inFRs without adversely affecting the binding properties of an antibody.

In certain embodiments, a monoclonal antibody binds to the ERG 42-66epitope and comprises a heavy chain that is at least about 70%, at leastabout 80%, at least about 90%, at least about 95% or at least about 98%identical to the amino acid sequence of the heavy chain variable domainof the 9FY antibody, and a light chain that is at least about 70%, atleast about 80%, at least about 90%, at least about 95% or at leastabout 98% identical to the amino acid sequence of the light chainvariable domain of the 9FY antibody. In other embodiments, a monoclonalantibody binds to the ERG 42-66 epitope and has six CDRs (H1, H2, H3,L1, L2, and L3) that are at least about 70%, at least about 80%, atleast about 90%, at least about 95% or at least about 98% identical tothe six CDRs (H1, H2, H3, L1, L2, and L3) of the heavy and light chainsequences of the 9FY antibody. In one embodiment, the monoclonalantibody binds to the ERG 42-66 epitope and comprises a light chainvariable domain identical to SEQ ID NO:2 except for 1, up to 2, up to 3,up to 4, up to 5, up to 6, up to 7, and in certain cases, up to 10 aminoacid substitutions in the CDR sequences. In another embodiment, themonoclonal antibody binds to the ERG 42-66 epitope and comprises a heavychain variable domain identical to SEQ ID NO:3 except for 1, up to 2, upto 3, up to 4, up to 5, up to 6, up to 7, and in certain cases, up to 10amino acid substitutions in the CDR sequences. The specific amino acidpositions that can be substituted in a CDR, as well as the donor aminoacid that can be substituted into those positions can be readilydetermined by one of skill in the art using known methods, such as thosedisclosed in published U.S. Application 2006/0099204, the disclosure ofwhich is hereby incorporated by reference in its entirety.

In certain embodiments, the monoclonal antibody is a humanized antibodythat binds to the ERG 42-66 epitope and comprises (a) a light chainvariable domain comprising the CDR1, CDR2, and CDR3 sequences of SEQ IDNO:2; (b) a heavy chain variable domain comprising the CDR1, CDR2, andCDR3 sequences of SEQ ID NO:3; and (c) the framework regions of thelight and heavy chain variable regions of a human antibody. Thehumanized antibody optionally further comprises light and heavy chainconstant regions of at least one human antibody.

In another embodiment, the monoclonal antibody is a chimeric antibodythat binds to the ERG 42-66 epitope and comprises a light chain variabledomain comprising the amino acid sequence of SEQ ID NO:2 and a heavychain variable domain comprising the amino acid sequence of SEQ ID NO:3.The chimeric antibody optionally further comprises light and heavy chainconstant regions of at least one human antibody. In another embodiment,the monoclonal antibody is a human antibody that binds to the ERG 42-66epitope.

In certain embodiments, the monoclonal antibody that binds to the ERG42-66 epitope has a dissociation constant (K_(D)) of about 280 nM orless. In other embodiments, the monoclonal antibody that binds to theERG 42-66 epitope detects ERG expressing carcinoma with a specificity ofgreater than 99%. In other embodiments, the monoclonal antibody thatbinds to the ERG 42-66 epitope does not cross react with the human FLi1protein. Cross reactivity can be measured using routine methods in theart, including, for example, a Western blot.

The antibodies provided in this disclosure that bind to the ERG 42-66epitope are optionally isolated.

It may also be desirable to modify the antibodies of the presentinvention to improve effector function, e.g., so as to enhanceantigen-dependent cell-mediated cytotoxicity (ADCC) and/or complementdependent cytotoxicity (CDC) of the antagonist. One or more amino acidsubstitutions or the introduction of cysteine in the Fc region may bemade, thereby improving internalization capability and/or increasedcomplement-mediated cell killing and ADCC. See Caron et al., J. Ex. Med.176:1191-1195 (1991) and Shopes, B. J. Immunol. 148:2918-2022 (1992),incorporated herein by reference in their entirety. An antibody fusionprotein may be prepared that has dual Fc regions with both enhancedcomplement lysis and ADCC capabilities. Typical Fc receptors that bindto an Fc region of an antibody (e.g., an IgG antibody) include, but arenot limited to, receptors of the FcγRI, FcγRII, and FcγRIII and FcRnsubclasses, including allelic variants and alternatively spliced formsof these receptors. Fc receptors are reviewed in Ravetch and Kinet,Annu. Rev. Immunol 9:457-92, 1991; Capel et al., Immunomethods 4:25-34,1994; and de Haas et al., J. Lab. Clin. Med. 126:330-41, 1995).

3. Nucleic Acids, Cloning and Expression Systems

The present disclosure further provides isolated nucleic acids encodingthe disclosed antibodies or portions thereof. The nucleic acids maycomprise DNA or RNA and may be wholly or partially synthetic orrecombinant. Reference to a nucleotide sequence as set out hereinencompasses a DNA molecule with the specified sequence, and encompassesa RNA molecule with the specified sequence in which U is substituted forT, unless context requires otherwise.

The nucleic acids provided herein encode at least one CDR (H1, H2, H3,L1, L2, and/or L3), a V_(L) domain (SEQ ID NO:2), and/or a V_(H) domain(SEQ ID NO:3) of the 9FY antibody.

The present disclosure also provides constructs in the form of plasmids,vectors, phagemids, transcription or expression cassettes which compriseat least one nucleic acid encoding a CDR, a V_(H) domain, and/or a V_(L)domain of the 9FY antibody. The disclosure further provides a host cellwhich comprises one or more constructs as above.

Also provided are methods of making the polypeptides encoded by thesenucleic acids. The method comprises expressing the encoded polypeptidefrom the encoding nucleic acid. Expression may be achieved by culturingunder appropriate conditions recombinant host cells containing thenucleic acid. Following production by expression a V_(H) or V_(L)domain, or specific binding member may be isolated and/or purified usingany suitable technique, then used as appropriate.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known in the art. For cells suitable forproducing antibodies, see Gene Expression Systems, Academic Press, eds.Fernandez et al., 1999. Any protein expression system compatible withthe invention may be used to produce the disclosed antibodies. Suitableexpression systems include transgenic animals described in GeneExpression Systems, Academic Press, eds. Fernandez et al., 1999.

Suitable vectors can be chosen or constructed, so that they containappropriate regulatory sequences, including promoter sequences,terminator sequences, polyadenylation sequences, enhancer sequences,marker genes and other sequences as appropriate.

A further aspect of the disclosure provides a host cell comprising anucleic acid as disclosed herein. A still further aspect provides amethod comprising introducing such nucleic acid into a host cell. Theintroduction may employ any available technique. For eukaryotic cells,suitable techniques may include calcium phosphate transfection,DEAE-Dextran, electroporation, liposome-mediated transfection andtransduction using retrovirus or other virus, e.g., vaccinia or, forinsect cells, baculovirus. For bacterial cells, suitable techniques mayinclude calcium chloride transformation, electroporation andtransfection using bacteriophage. The introduction of the nucleic acidinto the cells may be followed by causing or allowing expression fromthe nucleic acid, e.g., by culturing host cells under conditions forexpression of the gene.

4. Methods of Making Antibodies

Numerous methods known to those skilled in the art are available forobtaining antibodies or antigen-binding fragments thereof. For example,antibodies can be produced using recombinant DNA methods. See, e.g.,U.S. Pat. No. 4,816,567, EPO 8430268.0; EPO 85102665.8; EPO 85305604.2;PCT/GB 85/00392; EPO 85115311.4; PCT/US86/002269; and Japaneseapplication 85239543, the disclosures of which are incorporated hereinby reference in their entirety.

Monoclonal antibodies may also be produced by preparing immortalizedcell lines capable of producing antibodies having desired specificity.Such immortalized cell lines may be produced in a variety of ways.Conveniently, a small vertebrate, such as a mouse, is hyperimmunizedwith the desired immunogen. The vertebrate is then sacrificed, usuallyseveral days after the final immunization, the spleen cells removed, andthe spleen cells immortalized. The most common technique is fusion witha myeloma cell fusion partner, as first described by Kohler and Milstein(1975) Nature 256:495-497. Other techniques including EBVtransformation, transformation with bare DNA, e.g., oncogenes,retroviruses, etc., or any other method which provides for stablemaintenance of the cell line and production of monoclonal antibodies.Specific techniques for preparing monoclonal antibodies are described inAntibodies: A Laboratory Manual, Harlow and Lane, eds., Cold SpringHarbor Laboratory, 1988, the full disclosure of which is incorporatedherein by reference.

Immortalized cell lines can be screened using standard methods, such asenzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance(BIACORE™) analysis, to identify one or more hybridomas that produce anantibody that specifically binds with a specified antigen. Any form ofthe specified antigen may be used as the immunogen, e.g., recombinantantigen, naturally occurring forms, any variants or fragments thereof,as well as antigenic peptide thereof.

One exemplary method of making antibodies includes screening proteinexpression libraries, e.g., phage or ribosome display libraries. Phagedisplay is described, for example, in Ladner et al., U.S. Pat. No.5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al. (1991)Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597WO92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO92/01047; WO 92/09690; and WO 90/02809, the disclosures of which areincorporated herein by reference in their entirety.

In addition to the use of display libraries, the specified antigen canbe used to immunize a non-human animal, e.g., a rodent, e.g., a mouse,hamster, or rat. In one embodiment, the non-human animal includes atleast a part of a human immunoglobulin gene. For example, it is possibleto engineer mouse strains deficient in mouse antibody production withlarge fragments of the human Ig loci. Using the hybridoma technology,antigen-specific monoclonal antibodies derived from the genes with thedesired specificity may be produced and selected. See. e.g., XENOMOUSE™,Green et al. (1994) Nature Genetics 7:13-21, US 2003-0070185, WO96/34096, published Oct. 31, 1996, and PCT Application No.PCT/US96/05928, filed Apr. 29, 1996, the disclosures of which areincorporated herein by reference in their entirety.

In another embodiment, a monoclonal antibody is obtained from thenon-human animal, and then modified. For example, humanized,deimmunized, and chimeric antibodies may be produced using recombinantDNA techniques known in the art. A variety of approaches for makingchimeric antibodies have been described. See e.g., Morrison et al.,Proc. Natl. Acad. Sci. U.S.A. 81:6851, 1985; Takeda et al., Nature314:452, 1985, Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al.,U.S. Pat. No. 4,816,397; Tanaguchi et al., European Patent PublicationEP171496; European Patent Publication 0173494, United Kingdom Patent GB2177096B, the disclosures of which are incorporated herein by referencein their entirety. Humanizing an antibody involves transplanting thecombining-site of a nonhuman antibody onto a human antibody. This may beperformed by grafting the nonhuman CDRs onto human framework andoptionally human constant regions or by transplanting the entirenonhuman variable domains but hiding them with a human-like surface byreplacement of certain exposed residues. Details on creating a humanizedantibody are disclosed in U.S. Pat. No. 5,472,693, which is herebyincorporated by reference.

Humanized antibodies may also be produced, for example, using transgenicmice that express human heavy and light chain genes, but are incapableof expressing the endogenous mouse immunoglobulin heavy and light chaingenes. Winter describes an exemplary CDR-grafting method that may beused to prepare the humanized antibodies described herein (U.S. Pat. No.5,225,539, the disclosure of which is incorporated herein by referencein its entirety). All of the CDRs of a particular human antibody may bereplaced with at least a portion of a non-human CDR, or only some of theCDRs may be replaced with non-human CDRs. It is only necessary toreplace the number of CDRs required for binding of the humanizedantibody to a predetermined antigen.

Humanized antibodies or fragments thereof can be generated by replacingsequences of the Fv variable domain that are not directly involved inantigen binding with equivalent sequences from human Fv variabledomains. Exemplary methods for generating humanized antibodies orfragments thereof are provided by Morrison (1985) Science 229:1202-1207;by Oi et al. (1986) BioTechniques 4:214; and by U.S. Pat. Nos.5,585,089; 5,693,761; 5,693,762; 5,859,205; and 6,407,213, thedisclosures of which are incorporated herein by reference in theirentirety. Those methods include isolating, manipulating, and expressingthe nucleic acid sequences that encode all or part of immunoglobulin Fvvariable domains from at least one of a heavy or light chain. Suchnucleic acids may be obtained from a hybridoma producing an antibodyagainst a predetermined target, as described above, as well as fromother sources. The recombinant DNA encoding the humanized antibodymolecule can then be cloned into an appropriate expression vector.

In certain embodiments, a humanized antibody is optimized by theintroduction of conservative substitutions, consensus sequencesubstitutions, germline substitutions and/or backmutations. Such alteredimmunoglobulin molecules can be made by any of several techniques knownin the art, (e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80:7308-7312, 1983; Kozbor et al., Immunology Today, 4: 7279, 1983; Olssonet al., Meth. Enzymol., 92: 3-16, 1982), the disclosures of which areincorporated herein by reference in their entirety, and may be madeaccording to the teachings of PCT Publication WO92/06193 or EP 0239400,the disclosures of which are incorporated herein by reference in theirentirety. Typically, conservative substitutions are made. Often, but notexclusively, an amino acid common to a position in human germlineantibody sequences may be used. Human germline sequences, e.g., aredisclosed in Tomlinson, et al. (1992) J. Mol. Biol. 227:776-798; Cook,G. P. et al. (1995) Immunol. Today Vol. 16 (5): 237-242; Chothia, D. etal. (1992) J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995) EMBOJ. 14:4628-4638, the disclosures of which are incorporated herein byreference in their entirety. The V BASE directory provides acomprehensive directory of human immunoglobulin variable regionsequences (compiled by Tomlinson, I. A. et al. MRC Centre for ProteinEngineering, Cambridge, UK). These sequences can be used as a source ofhuman sequence, e.g., for framework regions and CDRs. Consensus humanframework regions can also be used, e.g., as described in U.S. Pat. No.6,300,064, the disclosure of which is incorporated herein by referencein its entirety.

Human antibodies can be generated using methods known in the art, suchas phage display technology. Phage display technology mimics themammalian immune system by cloning large libraries of antibody genes andselecting for binding to a desired target, such as the ERG 42-66epitope. The libraries used in phage display technology can be made fromseveral sources. For example, an immune library, created from humansexposed to a desired antigen through vaccination or disease, has highlevels of circulating antibodies to the antigen even when the library isrelatively small. As another example, a naïve library, made from mRNAisolated from non-immunized individuals, can be used repeatedly toisolate antibodies against a variety of antigens. As still anotherexample, a synthetic library, in which germline antibody gene segmentsare cloned and arranged combinatorially in vitro to reconstitute genesencoding complete V_(H) and V_(L) chains, has the advantage of producingantibodies with specificity to self-antigens. Semi-synthetic librariescan also be made by selecting one or more antibody frameworks andrandomizing sequences within the CDR loops.

In phage display technology, once a library is created, it is fused to asurface protein of phages, commonly pIII. In a process known as panning,phages displaying an antibody specific for the antigen of interest areenriched by selective adsorption onto immobilized antigen. Subsequently,the bound phage can be eluted from the surface and amplified throughinfection of E. coli cells.

Other modifications of phage display technology to generate humanantibodies are also known in the art. For example, antibodies can bedisplayed on the surfaces of microbial cells, such as E. coli andSaccharomyces cerevisaie, instead of on the surface of bacteriophages.In this case, screening can be performed by incubation with afluorescently tagged ligand in buffer. Cells that display the antibodiesthat bind to the ligand become fluorescently labeled and are isolated byfluorescence-activated cell sorting. Another modification, termedribosome display, relies on the formation of a ternary complex betweenribosomes, mRNA, and the polypeptide.

Another method known in the art to produce human antibodies is one thatuses transgenic mice. The native immunoglobulin repertoire in these micehas been replaced with human V-genes in the murine chromosome. The micecan be injected with a desired antigen and the resulting antibodies canbe recovered by cloning and screening an immune library, or byconventional hybridoma technology. These mice produce significant levelsof fully human antibodies that only differ in glycosylation patterns.

The anti-ERG antibodies described herein can be derivatized or linked toanother functional molecule (such as another peptide or protein (e.g., aFab fragment)). For example, the antibody can be functionally linked(e.g., by chemical coupling, genetic fusion, non-covalent association orotherwise) to at least one other molecular entity, such as anotherantibody (e.g., a bispecific or a multispecific antibody), toxin,radioisotope, cytotoxic or cytostatic agent, among others.

5. Methods of Use

The monoclonal antibodies described herein that bind to the ERG 42-66epitope can be used in a variety of research and medical applications.In one aspect, the disclosure provides a method of treating a malignancyor disease in a subject, comprising administering to said subject atherapeutically effective amount of a monoclonal antibody describedherein that binds to the ERG 42-66 epitope formulated in apharmaceutically acceptable vehicle, wherein the malignancy or diseaseis caused by an ERG fusion event (e.g., fusion between a TMPRSS2,SLC45A3, or NDRG1 gene promoter sequence and an ERG gene sequence orbetween a first gene sequence and an ERG gene sequence) and/or ERGoverexpression. In one embodiment, the malignancy or disease is prostatecancer, Ewing's sarcoma, acute myeloid leukemia, acute T-lymphoblasticleukemia, megakaryoblastic leukemia, colon cancer, or a disease of theendothelial cells, such as endothelial cancer. In another embodiment,the disease is Alzheimer's disease or Down's syndrome.

In addition, the monoclonal antibodies described herein that bind to theERG 42-66 epitope can be used to detect ERG proteins, including fusionproteins comprising all or part of an ERG polypeptide, such as an ERGprotein encoded by a TMPRSS2/ERG, SLC45A3/ERG, or NDRG1/ERG fusiontranscript, in a biological sample. In one embodiment, the methodcomprises contacting the monoclonal antibody described herein that bindsto the ERG 42-66 epitope with the biological sample and analyzing thebiological sample to detect binding of the monoclonal antibody to humanERG or the fusion protein in the biological sample. In one embodiment,the ERG polypeptide is ERG3. In another embodiment, the ERG proteinencoded by the TMPRSS2/ERG or SLC45A3/ERG fusion transcript is atruncated ERG3 protein. In one embodiment, the biological samplecomprises a tissue or a cell, such as a prostate tissue or a prostatecell. In other embodiments, the biological sample comprises a biologicalfluid, such as urine or blood, wherein the biological fluid containscancer cells, such as prostate cancer cells. In other embodiments, thebiological fluid is blood, serum, urine, saliva, sputum, or stool.

In another embodiment, the disclosure provides a method for detectingcancer or a disease in a patient, the method comprising contacting amonoclonal antibody described herein that binds to the ERG 42-66 epitopewith a biological sample obtained from the patient and analyzing thebiological sample, wherein binding of the antibody to cells in thebiological sample indicates the presence of cancer in the biologicalsample. In one embodiment, the cancer is prostate cancer. In anotherembodiment, the cancer is Ewing sarcoma, acute myeloid leukemia, acuteT-lymphoblastic leukemia, megakaryoblastic leukemia, colon cancer, orendothelial cancer. In one embodiment, the biological sample comprises atissue or a cell, such as a prostate tissue or a prostate cell. In otherembodiments, the biological sample comprises a biological fluid, such asurine or blood, wherein the biological fluid contains cancer cells, suchas prostate cancer cells. In other embodiments, the biological fluid isblood, serum, urine, saliva, sputum, or stool. In one embodiment, thecancer or disease is associated with an ERG fusion event (e.g., fusionbetween a TMPRSS2, SLC45A3, or NDRG1 gene promoter sequence and an ERGgene sequence) and/or ERG over expression. The binding of the antibodyto hematopoietic cells or endothelial cells in blood vessels orcapillaries is normal and typically does not indicate the presence ofcancer. However the detection of an ERG protein, including fusionproteins comprising all or part of an ERG polypeptide, in any cell otherthan a normal hematopoietic cell or a normal endothelial cell in bloodvessels or capillaries indicates the presence of cancer in the sample.

Also, as shown in the examples, the anti-ERG antibodies described hereincan be used to detect ERG-positive PIN. Patients exhibiting ERG-positiveprostatic intraepithelial neoplasia (PIN) likely also have ERG-positiveprostate cancer or are at high risk for developing ERG-positive prostatecancer. Thus, if ERG-positive PIN, but not ERG-positive prostate cancer,is detected in a patient, the patient should undergo additional biopsiesor more frequent biopsies to monitor the development of prostate cancerin the patient. Accordingly, the anti-ERG antibodies described hereincan be used to inform decisions about whether to perform additionalbiopsies or the frequency of future biopsies.

In yet another aspect, the monoclonal antibodies described herein thatbind to the ERG 42-66 epitope can be used to monitor the efficacy oftherapeutic regimens. Thus, one embodiment is directed to a method ofmonitoring hormone ablation therapy, the method comprising contacting amonoclonal antibody described herein that binds to the ERG 42-66 epitopewith a biological sample obtained from a patient who has receivedhormonal ablation therapy, and measuring the expression of human ERG ora fusion protein comprising all or part of a human ERG polypeptide inthe biological sample, wherein reduced ERG expression following androgenablation therapy indicates the hormone ablation therapy was effectiveand correlates with an increased survival time. The level of ERGexpression in the patient after hormonal ablation therapy can becompared to the level of ERG expression in the same patient beforecommencing hormonal ablation therapy or can be compared to astandardized control value, representing a heightened level of ERGexpression.

Also provided is a method for identifying a nucleic acid sequence, suchas a promoter sequence, or a polypeptide that interacts with an ERGpolypeptide. Nucleic acids and/or proteins identified by such methodsmay define new therapeutic targets in the ERG network. In oneembodiment, the method comprises incubating a sample comprising anucleic acid molecule or a polypeptide with an ERG polypeptide,incubating the sample and the ERG polypeptide with an antibody thatbinds to the 42-66 ERG epitope, and determining whether a complex formsbetween the nucleic acid molecule and the ERG polypeptide or between thepolypeptide and the ERG polypeptide, wherein detecting the formation ofa complex with the antibody indicates that the nucleic acid molecule orpolypeptide interacts with the ERG polypeptide. Such methods can becarried out using techniques that are conventional in the art,including, for example, immunoprecipitation assays.

Any appropriate label may be used in the methods and compositionsdescribed herein. A label is any molecule or composition bound to ananalyte, analyte analog, detector reagent, or binding partner (e.g.,antibody) that is detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical or chemical means.Examples of labels, including enzymes, colloidal gold particles, coloredlatex particles, have been disclosed (U.S. Pat. Nos. 4,275,149;4,313,734; 4,373,932; and 4,954,452, each incorporated by referenceherein). Additional examples of useful labels include, withoutlimitation, haptens (e.g., biotin, digoxigenin (DIG), dintrophenol(DNP), etc.), radioactive isotopes, co-factors, ligands,chemiluminescent or fluorescent agents, protein-adsorbed silverparticles, protein-adsorbed iron particles, protein-adsorbed copperparticles, protein-adsorbed selenium particles, protein-adsorbed sulphurparticles, protein-adsorbed tellurium particles, protein-adsorbed carbonparticles, and protein-coupled dye sacs. The attachment of a compound toa label can be through any means, including covalent bonds, adsorptionprocesses, hydrophobic and/or electrostatic bonds, as in chelates andthe like, or combinations of these bonds and interactions and/or mayinvolve a linking group.

Some labels, such as haptens, are typically coupled with detectionreagents for colorimetric detection. For example, biotin can be coupledto a streptavidin-HRP compound that is colorimetrically detected usingDAB. Those skilled in the art will appreciate the myriad label anddetection options which find utility as detection reagents useful incompositions and methods of the present invention.

In some embodiments, a molecule, such as an antibody (e.g., monoclonalantibody), is detected directly by conjugation with a detectable moiety.For example, monoclonal antibodies as described herein can be directlyconjugated to a detectable moiety, such as a fluorescent compound(including fluorescein, fluorescein isothiocyanate (FITC), rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors, ALEXA FLUOR® (Invitrogen, Carlsbad, Calif.) dyes, or thecyanine family of dyes (such as Cy-3 or Cy-5) and the like); abioluminescent compound (such as luciferase, green fluorescent protein(GFP), yellow fluorescent protein, etc.); an enzyme that produces adetectable reaction product (such as horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase, or glucose oxidaseand the like), or a radiolabel (such as ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, or ¹³¹I).

In some embodiments, a molecule, such as an antibody (e.g., monoclonalantibody), is indirectly detected. For example, secondary antibodies areraised against primary antibodies (e.g., a monoclonal ERG antibody asdescribed herein) as known to a skilled artisan, where the secondaryantibody is labeled for detection. The secondary antibody is conjugatedto a detectable moiety. For example, the secondary antibody can beconjugated to a reporter enzyme such as alkaline phosphatase (AP) orhorseradish peroxidase (HRP) for subsequent colorimetric detection. Insome embodiments, the secondary antibody is conjugated to a fluorescentmoiety as previously described (e.g., FITC, Cy dyes, ALEXA FLUOR®(Invitrogen, Carlsbad, Calif.) dyes, rhodamine, etc.). In someembodiments, a primary monoclonal ERG antibody in indirectly detectedwith a secondary antibody conjugated to a hapten, such as, biotin DNP,DIG, etc., which is further coupled to a detectable reagent moleculesuch as streptavidin-HRP, which is subsequently detectedcolorimetrically by reaction with 3,3′-Diaminobenzidine (DAB). A skilledartisan will appreciate the myriad of enzymatic and luminescentdetection methods utilized for visualization of protein-proteininteractions as described herein.

6. Formulations and Administration

The disclosure provides compositions comprising a monoclonal antibodydescribed herein that binds to the ERG 42-66 epitope. In certainembodiments, the compositions are suitable for pharmaceutical use andadministration to patients. These compositions comprise a monoclonalantibody described herein that binds to the ERG 42-66 epitope and apharmaceutically acceptable excipient. The compositions may also containother active compounds providing supplemental, additional, or enhancedtherapeutic functions. The pharmaceutical compositions may also beincluded in a container, pack, or dispenser together with instructionsfor administration. In one embodiment, the composition comprises amonoclonal antibody described herein that binds to the ERG 42-66 epitopefor treatment of a disease, such as Alzheimer's disease, or amalignancy, such as prostate cancer, Ewing's sarcoma, acute myeloidleukemia, acute T-lymphoblastic leukemia, megakaryoblastic leukemia,colon cancer or endothelial cancer.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Methods toaccomplish the administration are known to those of ordinary skill inthe art. This includes, for example, injections, by parenteral routessuch as intravenous, intravascular, intraarterial, subcutaneous,intramuscular, intratumor, intraperitoneal, intraventricular,intraepidural, or others as well as oral, nasal, ophthalmic, rectal, ortopical. Sustained release administration is also specificallycontemplated, by such means as depot injections or erodible implants.Localized delivery is particularly contemplated, by such means asdelivery via a catheter to one or more arteries, such as the renalartery or a vessel supplying a localized tumor.

In one embodiment a subject antibody is administered to a patient byintravenous, intramuscular or subcutaneous injection. An antibody may beadministered within a dose range between about 0.1 mg/kg to about 100mg/kg; between about 1 mg/kg to 75 mg/kg; or about 10 mg/kg to 50 mg/kg.The antibody may be administered, for example, by bolus injunction or byslow infusion. Slow infusion over a period of 30 minutes to 2 hours maybe used.

Toxicity and therapeutic efficacy of the composition can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Antibodies that exhibit large therapeutic indices may be less toxicand/or more therapeutically effective.

7. Kits

In some embodiments, ERG related molecules as described herein (e.g.,monoclonal antibody, polypeptide, etc.) are supplied in the form of akit useful, for example, for performing the methods of the presentinvention. In one embodiment, an appropriate amount of at least one ERGrelated molecule (e.g., monoclonal antibody, polypeptide, etc.) isprovided in one or more containers. In other embodiments, at least oneERG related molecule (e.g., monoclonal antibody, polypeptide, etc.) isprovided suspended in an aqueous solution or as a freeze-dried orlyophilized powder, for instance. The container(s) in which the at leastone ERG related molecule (e.g., monoclonal antibody, polypeptide, etc.)is supplied can be any conventional container that is capable of holdingthe supplied form, for instance, microfuge tubes, ampoules, or bottles.The amount of ERG related molecule (e.g., monoclonal antibody,polypeptide, etc.) supplied can be any appropriate amount.

In other embodiments, control slides upon which are mounted one or moretissue or cell preparations (e.g., biopsy, xenografts, cell pellets, orclotted cells) that may serve as positive and/or negative controls for aERG related molecule (e.g., monoclonal antibody, polypeptide, etc.) maybe provided in an appropriate and separate container.

Other kit embodiments include means for detection of the ERG relatedmolecule, such as secondary antibodies. In some such instances, thesecondary antibody is directly labeled with a detectable moiety (asdescribed elsewhere in this disclosure). In other instances, the primaryor secondary (or higher-order) antibody is conjugated to a hapten (suchas biotin, DNP, DIG, etc.), which is detectable by a detectably labeledcognate hapten-binding molecule (e.g., streptavidin (SA)-horse radishperoxidase, SA-alkaline phosphatase, SA-QDot® (Invitrogen, Carlsbad,Calif.), etc.). In some embodiments, the primary or secondary antibodyin conjugated with a fluorescent detection moiety (e.g., FITC,rhodamine, ALEXA FLUOR® (Invitrogen, Carlsbad, Calif.) dyes, Cydesignated fluorophores, etc.) Some kit embodiments may includecolorimetric reagents (e.g., DAB, AEC, etc.) in suitable containers tobe used in concert with primary or secondary (or higher-order)antibodies that are labeled with enzymes for the development of suchcolorimetric reagents.

In one embodiment, a kit includes instructional materials disclosingmethods of use of the kit contents (e.g., ERG related molecule) in adisclosed method. The instructional materials may be provided in anynumber of forms, including, but not limited to, written form (e.g.,hardcopy paper, etc.), in an electronic form (e.g., computer diskette orcompact disk) or may be visual (e.g., video files). The kits may alsoinclude additional components to facilitate the particular applicationfor which the kit is designed. Thus, for example, the kits mayadditionally include buffers and other reagents routinely used for thepractice of a particular method. Such kits and appropriate contents arewell known to those of skill in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

EXAMPLES Example 1. 9FY Antibody Generation

The hybridoma clone 9FY was obtained by immunizing Balb/C mice with achemically synthesized polypeptide having amino acids 42-66 of SEQ IDNO: 1 and a cysteine residue added at either the N- or C-terminus tokeep the —COOH free (unconjugated). The immunizing polypeptide wasconjugated to keyhole limpet hemocyanin (KLH) and injected into the micewith an adjuvant at three separate injection sites at three weekintervals. The first injection used Freund's complete as the adjuvant.The second and third injection used Freund's incomplete adjuvant. Serumbleeds were assessed for binding to the immunizing polypeptide using adirect ELISA screen. Mice with the highest titer were chosen for thefirst hybridoma fusion step. Eight positives clones were confirmed byELISA and supematants were analyzed by immunoblot assays using ERG3protein heterologously expressed in HEK-293 cells. Two of the cloneswere found to be positive in the immunoblot assay. One of the negativeclones from the secondary screen was retained as the negative control.The two positive clones and the negative control were further processedfor secondary cloning. One of the positive clones (9FY) that initiallyshowed the strongest positive activity survived the second cloning step.The 9FY clone was grown and injected into 10 mice for ascitesproduction. Ascites was produced and the 9FY antibody was purified overa Protein G column. The 9FY antibody sub-type was determined to be IgGfrom the purified fraction.

Example 2. 9FY Antibody Specifically Recognizes ERG Protein in ProstateCancer Cells

VCaP cells (Korenchuck et al., 2001, In Vivo, 15:163-68) are a humanprostate cancer cell line that over express a TMPRSS2/ERG fusionfrequently detected in human prostate tumors. In VCaP cells, endogenousERG gene transcription is controlled by the androgen inducible TMPRSS2promoter. LNCaP cells are a human prostate cancer cell line that do notharbor a TMPRSS2/ERG fusion and do not express detectable levels of ERG.

1. Western Blots

LNCaP (ATCC #CRL-1740) and VCaP cells (ATCC, #CRL-2876) were grown inRPMI-1640 (ATCC; #30-2001) and DMEM medium (ATCC; #30-2002),respectively, supplemented with 10% fetal bovine serum (ATCC; #30-2020)and 2 mM glutamine. LNCaP and VCaP cells (2×10⁶) were seeded onto 10 cmdishes and maintained for five days and three days, respectively, inmedia with 10% charcoal-stripped fetal bovine serum (c-FBS; #100119Gemini Bio-Products, Calabasas, Calif.). For androgen induction, thecells were subsequently grown in fresh media with c-FBS supplementedwith 0.1 nM R1881 or 1 nM R1881 for another 48 hours. Cells wereharvested and analyzed by Western blots and microscopy.

In Western blot experiments, the 9FY antibody recognizes full lengthprotein products (approximately 50-52 kDa) encoded by TMPRSS2/ERG2 andTMPRSS2/ERG3 in response to androgen (R1881) induction (FIG. 1A). The9FY antibody did not recognize ERG in LNCaP cells, which do not harbor aTMPRSS2/ERG fusion transcript.

To evaluate the specificity of the 9FY antibody recognition, syntheticandrogen (R1881) induced VCaP cells and LNCaP cells were transfectedwith ERG-specific small inhibitory RNA (ERG siRNA) (Sun et al., 2008)and/or a non-targeting (“NT”), control oligonucleotide. The ERG siRNA istargeted to a 19 base pair region in exon 11 and is predicted to inhibitall known ERG splice variants in prostate cells (Sun et al., 2008).Tubulin was used as a loading control in the immunoblot assay.

VCaP cell lysates were prepared in M-PER® mammalian protein extractionreagent (Cat #78501, Thermo, Rockford, Ill.) supplemented with proteaseand phosphatase inhibitor cocktails (Cat #P2850 & P5726, Sigma, St.Louis, Mo.), from VCaP cells transfected with NT or ERG siRNAoligonucleotides (Dharmacon, Lafayette, Colo.) and incubated for fourdays. Cells were transfected with 50 nM NT oligonucleotides, 25 nM NTand 25 nM ERG siRNA oligonucleotides or 50 nM ERG siRNA oligonucleotides(lane 3). 25 jtg of cell lysates were loaded in each lane, separated onNUPAGE® SDS gel (Cat #NP0335, Invitrogen, Carlsbad, Calif.), transferredonto PVDF membranes and Western blotted with 1:10,000 9FY antibody.Identical gels were transferred onto PVDF membranes (Cat #LC2005,Invitrogen, Carlsbad, Calif.) and probed with 1:1,000 anti-SLC45A3antibody (Cat M361529-2), 1:3,000 anti-PSA antibody (Cat # A056201-2,both from DAKO, Carpinteria, Calif.) and 1:1000 anti-alpha-tubulin (Cat#Sc-5286, Santa Cruz, Calif.).

LNCaP cell lysates were prepared from cells that have been grown inRPMI1640 supplemented with 10% charcoal stripped serum for five days andmaintained further in fresh R1881 free medium or stimulated with 0.1 nMor 1 nM R1881 for 48 hours. Similarly, VCaP cell lysates were preparedfrom cells that have been grown in DMEM supplemented with charcoalstripped serum for three days and maintained further in fresh R1881 freemedium or stimulated with 0.1 nM or 1 nM R1881 for 48 hours. 25 μg ofcell lysates were loaded in each lane, separated on NUPAGE® (Invitrogen,Carlsbad, Calif.) SDS gel, transferred onto PVDF membranes and Westernblotted with 1:10,000 9FY antibody. Identical gels were transferred ontoPVDF membranes and probed with 1:1,000 anti-SLC45A3 antibody, 1:3,000anti-PSA antibody and 1:1000 anti-alpha-tubulin as described above.VCaP, COLO320, MOLT-4 and KG-1 cells were lysed in M-PER® mammalianprotein extraction reagent, their protein concentration determined. 25μg of cell lysates were loaded in each lane, separated on NUPAGE®(Invitrogen, Carlsbad, Calif.) SDS gel, transferred onto PVDF membranesand Western blotted with 1:10,000 9FY antibody as described above.

Consistent with prior results, significant reduction of the endogenousERG protein levels were detected with the 9FY antibody (1:10,000dilution) in response to ERG siRNA treatment (FIG. 1B). As an additionalcontrol, we also examined the expression response of negativelyregulated downstream target genes of ERG, prostein (SLC45A3) andprostate specific antigen, PSA (KLK3) (Sun et al., 2008). As expected,marked upregulation of prostein and PSA was evident in VCaP cells inresponse to ERG siRNA (FIG. 1A and FIG. 1B, bottom panels).

2. ELISA

In an ELISA assay, we assessed the sensitivity and specificity of the9FY antibody measuring the titer of various concentrations of theimmunizing polypeptide (i.e. amino acids 42-66 of SEQ ID NO: 1). Fordirect ELISA, plates were first coated with 100 μl of ERG3 antigen at 1ng/ml, 10 ng/ml, 100 ng/ml and 1 μg/ml and incubated for 16 h at 4° C.on a shaker before washing the plate five times with PBSt (300 μl/well).Biotin-conjugated detecting 9FY antibody diluted in SuperBlock B wasadded at 74 μg/100 μl/well and the plates were incubated for 1 h on ashaker (this and all subsequent steps are performed at roomtemperature). The plate was washed with PBSt (300 μl/well). NextStreptAvidin-horse radish peroxidase (HRP) in PBS (100 μl/well) wasadded and incubated for 30 minutes on a shaker at room temperature. Theplate was then washed with PBSt (300 μl/well), before adding TMB at 100μl/well and allowed to develop for 5 min on a shaker before Stopsolution were added at 50 μl/well. Absorbance was measured at 450 nm ona MULTISKAN® Ascent (Thermo Scientific, Waltham, Mass.) ELISA platereader according to the manufacturer's recommendation and the absorbancewere plotted against antibody dilution. For indirect ELISA, plates werefirst coated with 1001l (570 μg)/well of C-epitope anti-ERG antibody andincubated for 16 h at 4° C. SuperBlock (Cat.# PI-37515, ThermoScientific, Rockford Ill.) was added at 300 μl/well and incubate for 1 h(this and all subsequent steps are performed at room temperature) on ashaker. 200 ng ERG3 antigen were added at (100 μl/well) and incubatedfor 2 hours on a shaker before the plate was washed with PBSt (300μl/well). Biotin-conjugated detecting 9FY antibody diluted in SuperBlockB was added at 74 μg/100 μl/well and the plates were incubated for 1 hon a shaker. The plate was washed with PBSt (300 μl/well) andStreptAvidin-HRP in PBS (100 μl/well) were next added and incubated for0.5-h on a shaker (room temperature). The plate was washed with PBSt(300 μl/well) again. Then tetramethylbenzidine (TMB) was added at 100ul/well and allowed to develop for 5 min on a shaker before Stopsolution was added at 50 μl/well. Absorbance was measured at 450 nm on aMULTISKAN® Ascent (Thermo Scientific, Waltham, Mass.) ELISA plate readeraccording to the manufacturer's recommendation and the absorbance wasplotted against antibody dilution.

The 9FY antibody recognized the immunizing polypeptide corresponding toamino acids 42-66 of SEQ ID NO: 1. Antibody titration and competitiveinhibition assay revealed that the immunizing polypeptide, but not twocontrol polypeptides, competes for the 9FY antibody binding against theERG3 protein (FIG. 2A) and that purified 9FY antibody can detect in the5000-20000× dilution range (FIG. 2B).

3. In Vivo Chromatin Immunoprecipitation Assay

To further assess the specificity of the 9FY antibody, we tested therecruitment of endogenous ERG to previously defined gene regulatoryelements by in vivo chromatin immunoprecipitation (ChIP) assay (Sun etal., 2008). Expression of ERG protein encoded by a TMPRSS2/ERG fusiontranscript was induced by R1881 in VCaP cells, and the cells weretransfected by either ERG siRNA to knock-down ERG or by controlNon-targeting (NT) siRNA. The cells were processed for ChIP assay andthe chromatin was immunoprecipitated using the 9FY antibody. In theabsence of ERG siRNA, endogenous ERG protein is recruited to target genesequences. In contrast, robust reduction of the ERG protein binding tothe HPGDH, C-MYC, prostein (SLC45A3) and PSAIKLK3 gene regulatoryregions (FIG. 3) was observed in response to ERG knockdown. This resultis consistent with the specific immunoprecipitation of ERG-boundchromatin by the antibody.

4. Immunofluorescence (IF)

The 9FY antibody was further evaluated in VCaP cells by the IF method(FIG. 4). VCaP cells were transfected with either NT- or ERG-siRNAoligonucleotides, the growth media were replaced with fresh DMEMsupplemented with 10% charcoal stripped serum and 0.1 nM R1881 andfurther incubated for 48 hours. For immunostaining, cells were firstfixed in fresh 4% Formaldehyde in phosphate buffered saline (PBS) andpermeabilized in PBS-T (PBS+0.1% TritonX-100) before being centrifugedonto glass slides with a CYTOSPIN® 4 (Thermo Scientific, Waltham, Mass.)centrifuge. Cells were then blocked in PBS-NT20 (PBS supplemented with1% normal horse serum (Cat #S-2000, Vector laboratories, Burlingame,Calif.) and 0.1% Tween-20. Cells were then incubated with primaryantibody against SLC45A3 or PSA (both from DAKO, Carpinteria, Calif.),diluted in PBS-NT20, at room temperature. Cells were washed withPBS-NT20 before goat anti-mouse ALEXA FLUOR® 594 (Invitrogen, Carlsbad,Calif.; Cat #A11302), goat anti-rabbit ALEXA FLUOR® 488 (Invitrogen,Carlsbad, Calif.; Cat #A11304) secondary antibodies and DAPI wereapplied. Cells were washed and mounted with FLUOROMOUNT G™(SouthemBiotech, Birmingham, Ala.) Images were captured using a 40×/0.65N-Plan objective on a Leica DMIRE2 inverted microscope with a QlmagingRetiga-EX CCD camera (Burnaby, BC, Canada), operated by OpenLab software(Improvision, Lexington, Mass.). Images were converted into color andmerged by using Photoshop (Adobe). For peptide competition, thecompetitive immunizing polypeptide (amino acids 42-66 of SEQ ID NO: 1)and non competitive, control ERG polypeptides were diluted from a stocksolution and mixed with the antibody at an excess of 2000-fold in molarconcentration over the antibody in the final concentration. Theantibody-polypeptide mixes were incubated on ice for 30 min beforeapplying to tissue specimen as described.

The 9FY antibody specifically recognized ERG protein as shown by nuclearstaining characteristic of the transcription factor function of ERG.VCaP cells were transfected with siRNA that efficiently knocks down ERGas it was previously shown (Sun et al., 2008). Indeed, robust reductionof endogenous ERG protein levels was observed in response to ERG siRNAtreatment compared to cells treated with control no-targeting siRNA. ERGis a repressor of prostate differentiation marker genes (Sun et al.,2008; Tomlins et al., 2008). Thus, elevation of PSA levels is consistentwith efficient ERG knockdown (FIG. 4A). To establish the recognitionspecificity of the 9FY antibody, the antibody was competed with a 2000×excess of the competitive immunizing polypeptide prior to the IF assayon VCaP cells. A 2000× fold molar excess of the immunizing polypeptide(Competing peptide) was sufficient to eliminate the IF signal in a 9FYantibody-specific manner. By contrast, control ERG polypeptides(Non-competing peptide) failed to compete with the 9FY antibody (FIG.4B). Cytoplasmic Prostate Specific Antigen (PSA) staining is notaffected by the immunizing polypeptide or the control polypeptide.Hormone inducible cytoplasmic expression of PSA was used as a positivecontrol for androgenic activation of ERG and PSA gene expressions (FIG.4C). In LNCaP cells that neither harbor TMPRSS2/ERG fusions nor expressdetectable levels of endogenous ERG, the 9FY antibody did not recognizeERG. PSA protein expression in response to R1881 is easily detectable inLNCaP cells and is consistent with the androgen-inducible expression ofPSA. Towards defining the concentration range for 9FY antibodyspecificity we found that ERG recognition by the 9FY antibody meets thecriteria even in the concentration range from 1:5,000 to 1:20,000dilution of 3.7 mg/ml stock (FIG. 4D). In the absence of 9FY antibody,the secondary antibody fails to detect any epitopes in VCaP cells. Thisresult excluded the possibility of non-specific staining of backgroundproteins in the IF experiments (FIG. 4E).

Example 3. Immunohistochemical Staining in FFPE Specimens

The 9FY antibody was also tested in formalin-fixed paraffin embedded(FFPE) human prostate specimens. Radical prostatectomy specimens werefixed in formalin and embedded as whole mounts in paraffin. Eachprostate was sectioned at 0.22 cm intervals in a transverse planeperpendicular to the long axis of the posterior surface of the prostateand completely embedded as whole mounts. The sections were analyzed forimmunohistochemistry (IHC) staining on four-micron sections of thewhole-mounted blocks.

The tissue sections for IHC were prepared as described previously(Furusato et al., 2007). Slides were incubated with 1:1200 9FY antibody.VECTOR® VIP (Vector Laboratories, Burlingame, Calif.) (purple) was usedas chromogen substrate and the slides were counterstained withhematoxylin. IHC revealed nuclear staining of prostate tumor cells thatis consistent with the sub-cellular localization of the ERGtranscription factor (FIGS. 5A-5D). In contrast, 9FY antibody stainsneither normal epithelia nor stroma in the human prostate. Endothelialcells of vasculature showed staining with 9FY antibody that isconsistent with the established constitutive expression of wild-type ERGin endothelial cells (Birdsey et al., 2008; McLaughlin et al., 1999;Rainis et al., 2005). Thus, endothelial cell staining can be used bypathologists as an internal reference when tumor epithelial IHCreactivity is observed with the 9FY antibody. Evaluation of whole mountspecimens from 150 plus prostate cancer patients has confirmed thespecificity of the 9FY antibody.

Example 4. Detection of ERG Protein in Other Cancer Cell Lines

Having established the specificity of the 9FY antibody in the VCaPprostate cancer cell line, we further evaluated this antibody for thedetection of ERG protein in cell lines that were previously described toexpress high levels of ERG RNAs, including COLO 320 (colon cancer (Quinnet al., 1979)), KG1 (acute myeloid leukemia, (Koeffler and Golde, 1978))and MOLT4 (human acute T lymphoblast leukemia (Minowada et al., 1972)).Colo320 (ATCC # CCL-220.1) and MOLT-4 (ATCC # CRL-1582) cells were grownin RPMI-1640 supplemented with 10% FBS. KG-1 cells were grown in Iscovemodified DMEM (Cat#12440-053, Invitrogen, Carlsbad, Calif.) supplementedwith 10% FBS. Cells were harvested and analyzed by Western blots andmicroscopy as described above. Immunoblot assay of COLO 320 (coloncancer (Quinn et al., 1979)), KG1 (acute myeloid leukemia, (Koeffler andGolde, 1978)) and MOLT4 (human acute T lymphoblast leukemia (Minowada etal., 1972)) with the 9FY antibody detected one predominant proteinspecies (FIG. 6), indicating the usefulness of this antibody indetecting and evaluating ERG proteins in human cancers other thanprostate cancer.

Example 5. Specificity of the 9FY Antibody

1. Western Blots

Using Western blots, the specificity of the 9FY antibody was compared tocommercially available polyclonal anti-ERG antibodies (C-20 (sc-353) orH95 (sc-28680)) using cell lysates from HEK293 cells transfected with aTMPRSS2/ERG3 fusion transcript. The 9FY antibody was used at 1:5000; C20(sc-353) (Santa Cruz Biotechnology, Inc, Santa Cruz, Calif.) and H95(sc-28680) (Santa Cruz Biotechnology, Inc, Santa Cruz, Calif.) were usedat 1:1000 dilutions. The 9FY antibody detects the exogenously expressedERG protein encoded by the TMPRSS2/ERG3 fusion transcript (52 kDa) as asingle species in Western blots of TMPRSS2/ERG3 transfected HEK293cells, highlighting the specificity of the 9FY antibody (FIG. 7). On theother hand, commercially available anti-ERG polyclonal antibodies reactwith multiple proteins in identical blots. Specifically, thecommercially available polyclonal C20 antibodies (sc-353) and polyclonalH95 antibodies (sc-28680) lacked the specificity of the 9FY antibody, asevidenced by the multiple bands in the Western blots obtained with thosepolyclonal antibodies (FIG. 7). According to the available productliterature, C20 recognizes an epitope mapping to the C-terminus of humanERG1, ERG2 and ERG3, while H95 recognizes an epitope mapping to aminoacids 26-120 of human ERG2 and ERG3 (Owczarek et al 2004).

The specificity of the polyclonal H95 antibodies was further analyzedusing cell lysates from LNCaP and VCaP cells. Lysates were prepared fromLNCaP and VCaP cells treated with or without the R1881 androgen ortransfected with Non Targeting (NT) or ERG siRNA oligonucleotides, asdescribed above. Notably, the H95 antibodies detected multiplenon-specific bands in LNCaP and VCaP cell lysates and questionabledetection of the target, a 52 kDa, truncated ERG polypeptide encoded bya TMPRSS2/ERG fusion transcript (compare FIGS. 9A/B with FIGS. 1A/B).

The 9FY antibody has also been used to detect an ERG oncoprotein encodedby the TMPRSS2/ERG8 fusion transcript. HEK-293 cells were grown in DMEMmedium, supplemented with 10% fetal bovine serum and 2 mM glutamine.HEK-293 cells (2×10⁶) were seeded onto 10 cm dishes and maintained fortwo days, before transfection with 4 μg of plasmid DNA pIRES-EGFP(CMV),pIRES-EGFP(CMV)-TMPRSS2-ERG3, pIRES-EGFP(CMV)-ERG3 orpIRES-EGFP(CMV)-TMPRSS2-ERG8. LIPOFECTAMINE™ 2000 (Invitrogen, Carlsbad,Calif.; Cat#11668-027) was used for transfection, and 48 hours latercells were harvested and total cell lysates equivalent to 4 or 20 gig ofprotein were analyzed by Western blotting using the 9FY antibody at1:5000 dilution. The 9FY antibody recognized predominant protein bandsconsistent with the predicted sizes of the wild type ERG3 protein (53kDa), the TMPRSS2/ERG3 encoded oncoprotein (50-52 kDa), and theTMPRSS2/ERG8 encoded protein (37 kDa) (FIG. 13).

The immunizing polypeptide showed no significant homology with proteinsbelonging to the human ETS family of proteins, except for a segment ofthe human FLi1 protein (Kubo et al., 2003) having 48% sequence identitywith the immunizing peptide. Therefore, the 9FY antibody was tested tosee if it can discriminate between human ERG3 and the human FLi1 proteinusing Western blot assays. In this assay, transiently expressed humanERG3 (encoded by TMPRSS2/ERG3 construct) or human FLi1 protein wasevaluated with the 9FY antibody. To assure that comparable amount of thehuman FLi1 protein was present in the assay both ERG3 and human FLi1proteins were expressed with C-terminal and with N-terminal FLAG tags,respectively. The 9FY antibody recognized only ERG3 protein and showedno immuno-reactivity with the human FLi1 protein.

2. Immunofluoresence (IF) Staining

The specificity of the C20 and H95 antibodies were further analyzedusing IF assays. Whereas the 9FY antibody exhibited nuclear staining ofprostate tumor cells (FIG. 4) in IF assays, polyclonal C20 (sc-353)antibodies recognize not only nuclear ERG protein but also non-specificcytoplasmic proteins (FIG. 9A, column 1) in VCaP cells. ERG knockdownwith ERG siRNA did not completely eliminate nuclear ERG and non-specificcytoplasmic staining (FIG. 9A, column 2) of the C-20 antibodies. The C20(sc-353) antibodies recognize ERG3 overexpressed in LNCaP cells byinfection with Ad-ERG3Adenoviral constructs (FIG. 9A, column 6) but thestaining was still detected after knockdown with ERG siRNA (FIG. 9A,column 7). Negative control experiments for siRNA and adenoviralinfection are shown in FIG. 9A, columns 3, 4 and 5.

The C20 (sc-353) antibodies recognize nuclear ERG protein in VCaP cells(FIG. 9B, column 1, upper panel). However, knockdown with ERG siRNA didnot eliminate nuclear ERG staining but rather resulted in increasedbackground (FIG. 9B, column 1, lower panel). In FIGS. 9A and 9B, ERG isstained by primary C20 (sc-353) antibodies followed by goat anti-rabbitALEXA FLUOR® 488 (Invitrogen, Carlsbad, Calif.) secondary antibody(green). In FIG. 9A, cells were counter stained with ALEXA FLUOR® 594tagged phalloidin (Invitrogen, Carlsbad, Calif.; Cat# A12381) to outlinethe nuclear structure. In FIG. 9B, the androgen inducible SLC45A3 thatis negatively controlled by ERG is stained by mouse monoclonalanti-SLC45A3 followed by goat anti-mouse ALEXA FLUOR® 594 (Invitrogen,Carlsbad, Calif.) secondary antibody (red). DNA is stained by DAPI(blue).

The H95 (sc-28680) antibodies, like the C-20 antibodies, stained nuclearproteins and non-specific cytoplasmic proteins (FIG. 10, column 1) inVCaP cells. In addition, ERG knockdown with ERG siRNA did not show anysignificant difference in staining (FIG. 10, column 2) in VCaP cells.ERG3 overexpressed in LNCaP cells by infection with Ad-ERG3 adenoviralconstructs was detected by H95 (sc-28680) (FIG. 10, column 6) but thestaining was not affected by knockdown with ERG siRNA (FIG. 10, column7). Moreover, in a negative control experiment for siRNA and adenoviralinfection (FIG. 10, columns 3, 4 and 5) in LNCaP cells (that do notharbor TMPRSS2/ERG fusion or express detectable levels of endogenousERG), the H95 (sc-28680) antibodies show high levels of non-specificbackground staining. In FIG. 10, ERG is stained by primary H95(sc-28680) followed by goat anti-rabbit ALEXA FLUOR® 488 (Invitrogen,Carlsbad, Calif.) secondary antibody (green). Cells were counter stainedwith ALEXA FLUOR® 594 (Invitrogen, Carlsbad, Calif.) tagged phalloidinto outline the nuclear structure. DNA is stained by DAPI (blue).

A third commercially available ERG polyclonal antibody, R-20 (sc-18136)(Santa Cruz Biotechnology, Inc, Santa Cruz, Calif.), was also analyzedby IF. According to the product literature available for R-20, it isrecommended for detecting ERG3 and binds an epitope that maps to aninternal region of human ERG3. ERG-3 R20 (sc-18136) exhibits nuclearstaining, as well as non-specific cytoplasmic staining (FIG. 11, column1). ERG knockdown with ERG siRNA reduced nuclear staining but did notcome close to eliminating the nuclear staining (FIG. 11, column 2), aswas observed with the 9FY antibody (FIG. 4A). ERG3 over-expressed inLNCaP cells by infection with Ad-ERG3 adenoviral constructs was detectedby R20 (sc-18136) (FIG. 11, column 6) but was still detected afterknockdown with ERG siRNA (FIG. 11, column 7). The R20 (sc-18136)antibodies also show high levels of background staining in negativecontrol experiment for siRNA and adenoviral infection (FIG. 11, columns3, 4 and 5). In FIG. 11, ERG is stained by primary R20 (sc-18136)antibodies followed by donkey anti-goat ALEXA FLUOR® 488 (Invitrogen,Carlsbad, Calif.) secondary antibody (green). Cells were counter stainedwith ALEXA FLUOR® 594 (Invitrogen, Carlsbad, Calif.) tagged phalloidinto outline the nuclear structure. DNA was stained by DAPI (blue).

Another IF assay was conducted to assess the ability of H95 and 9FY tocompetitively inhibit the binding of the other antibody to ERG. Theresults of this competitive IF assay confirm that the 9FY antibodyexclusively stains the nucleus of VCaP cells with high affinity. Incontrast, H-95 stains unknown protein(s) in the cytoplasm and nucleus ofVCaP cells. In head-to-head cross-competition, 10× excess of the 9FYantibody (2 μg/ml) inhibits the nuclear staining of H-95, however,non-specific cytoplasmic staining by H95 remains (FIG. 12, column 1). Incontrast, the 9FY antibody at (0.2 μg/ml) still shows distinct nuclearstaining in the presence of 10-fold excess of H-95 (2 μg/ml) (FIG. 12,column 5). At equal concentration of antibodies (0.4 μg/ml) the 9FYantibody shows stronger, highly specific and distinct nuclear stainingas compared to H95 (FIG. 12, column 3).

Consistent with the other antibody studies discussed above, theseresults show that the 9FY antibody recognizes a single protein in thenucleus of VCaP cells. In contrast, the H-95 polyclonal antibodies reactwith multiple undefined proteins in both the cytoplasm and nucleus.Furthermore, the 9FY antibody shows higher affinity when compared toH95.

3. Immunohistochemistry Staining

Using immunohistochemistry staining, the specificity of the 9FY antibodywas compared to a commercially available polyclonal anti-ERG antibodycalled C20 (sc 353) (Santa Cruz Biotechnology, Inc, Santa Cruz, Calif.).Because the 9FY antibody specifically recognizes human as well as mouseERG proteins, ERG specificity was assessed in formalin fixed paraffinembedded (FFPE) tissues from developing and adult mice.

Collection of Mouse Embryos and Adult Tissues.

Mouse embryos at 9.5, 14.5 and 17.5 days after mating were collectedfrom pregnant mice and fixed in 4% paraformaldehyde for 12-24 hours.Tissues from adult male mouse were collected and fixed in 4%paraformaldehyde for 24 hours. The tissues were processed for paraffinembedding and using microtome 4 μm sections were cut on to superfrostdouble plus slides

Immunohistochemistry (IHC) for ERG.

Following deparaffinization, 4 μm sections were dehydrated and blockedin 0.6% hydrogen peroxide in methanol for 20 min. Sections wereprocessed for antigen retrieval in 0.1M citrate buffer for 30 min in amicrowave followed by 30 min of cooling. The IHC procedure wascarried-out using Mouse-To-Mouse IHC Detection System Kit (MilliporeInc), according to the manufacturer's instruction. Briefly, sectionswere blocked with Pre-antibody Blocking solution for 10 minutes. Theblocking solution was removed, and the sections were incubated witheither the 9FY antibody or the commercially available C20 (sc 353) ERGantibody at a dilution of 1:1000 overnight at 4° C. After theincubation, the slides were washed with PBS for 5 minutes. Slides wereincubated with Post-antibody Blocking solution for 10 minutes and washedtwice with PBS for 5 minutes each. Slides were incubated with theready-to-use Poly-HRP-Anti-Mouse/Rabbit IgG for 10 minutes, washed withPBS for 5 minutes, two times and the color was developed with DABreagent. Sections were then counterstained in hematoxylin for 1 min,dehydrated, cleared, mounted and photographed.

Using the 9FY antibody, ERG was detected only in wild type mouseprostate endothelial cells and was not detected in wild type mouseprostate epithelium. FIG. 14A (Solid arrows mark prostate glands anddashed arrows indicate endothelial cells). On the other hand, thecommercially available C20 (sc 353) ERG antibody was non-specific forERG, staining both prostate endothelial and epithelial cells in tissuesobtained from wild type mice. FIG. 14B. As expected, in transgenic miceexpressing ERG, both antibodies detected ERG in prostate endothelial andepithelial cells. FIGS. 14C-D. However, the commercial C20 (sc 353) ERGantibody stains wild type and ERG-positive transgenic tissues with thesame intensity. FIGS. 14B and D.

The IHC analysis also revealed ERG expression in hematopoietic cells,endothelial cells, and in blood vessels and capillaries of variousembryonic tissues (data not shown). Similarly, expression of ERG inadult mouse tissues was detected in hematopoietic cells, endothelialcells in blood vessels and capillaries (data not shown). It is alreadyknown that subsets of AML and Ewing Sarcoma exhibit elevated ERGexpression. With the availability of an ERG-specific antibody, ERGalterations in pathologic conditions of hematopoietic cells, endothelialcells in blood vessels and capillaries in various embryonic tissues canbe pursued effectively. Interestingly, ERG is not detectable in normalepithelial cells in tissues, including prostate glands. Therefore, thedetection of ERG in any tissue other than hematopoietic cells, orendothelial cells in blood vessels and capillaries is an indication ofan abnormality and/or pathologic condition, such as, cancer.

Example 6. Sequencing of the 9FY Antibody Variable Domains

RNA was extracted from the 9FY hybridoma cell line and reversetranscribed into cDNA using an oligo d(T) primer specific for messengerRNA. The cDNA product was used as a template for PCR using primersspecific to murine antibody heavy and light chain sequences.

The PCR products were cloned into a standard sequencing vector andpositive colonies identified by PCR. These colonies were cultured andplasmids miniprepped for sequencing. Sequencing was performed using thedye termination method and analyzed on an ABI3130 genetic analyzer. Aconsensus sequence for the heavy and light chain variable domains wasdetermined by alignment with the program AlignX.

Based on this alignment, the light chain variable domain of the 9FYantibody was determined to comprise the following amino acid sequence(CDR1 (L1), CDR2 (L2), and CDR3 (L3) are underlined):

(SEQ ID NO: 2) ENVLTQSPAIMSASLGEKVTLSCRASSSVYYMFWYQQKSDASPKLWIYYTSNLAPGVPARFSGSGSGNSYSLTISSVEGEDAATYYCLQFSTSPWTFGGG TKLEIKR

And the heavy chain variable domain of the 9FY antibody was determinedto comprise the following amino acid sequence (CDR1 (H1), CDR2 (H2), andCDR3 (H3) are underlined):

(SEQ ID NO: 3) QIQLVQSGPDLKKPGETVKISCKASGYTFTNYGINWVKQAPGKGFKWMGWIDTYTGEPTYVDDFKGRFVFSLETSASTAYLQINNLKNEDTATYFCVRKR AYDYEIYWGQGTPLTVSS

Example 7. Analysis of ERG Oncoprotein and TMPRSS2/ERG Fusion Status inProstate Cancer

To assess the relationship between ERG oncoprotein staining in tumorspecimens and TMPRSS2-ERG fusion status, a comparative analysis wasperformed on consecutive tissue sections of the ERG oncoprotein positiveor ERG oncoprotein negative FFPE specimens for the detection ofTMPRSS2-ERG mRNA and overall ERG mRNA.

Prostate Specimens.

Prostate specimens for this analysis and for mapping ERG expression inmulti-focal prostate cancer (Example 8) were obtained and prepared asfollows. Radical prostatectomy specimens of 132 patients enrolled in theCenter for Prostate Disease Research program were obtained bypathologists within 30 minutes after the specimen was surgicallyremoved. Radical prostatectomy specimens were fixed in formalin,embedded as whole mounts in paraffin, sectioned at 0.22 cm intervals ina transverse plane perpendicular to the long axis of the posteriorsurface of the prostate, and completely embedded as whole mounts,according to our Armed Forces Institute of Pathology (AFIP) protocol (B.Furusato et al., 2008). From each patient, one whole mount cross sectioncontaining one to four tumors (mostly two foci) was selected and tumorsrepresented different grades and stages. Each tumor was separatelydiagnosed in the prostatectomy specimens and slices with more than onetumor focus represented separate tumors.

Immunohistochemistry for ERG.

Following deparaffinization, 4 μm sections were dehydrated and blockedin 0.6% hydrogen peroxide in methanol for 20 min. Sections wereprocessed for antigen retrieval in EDTA (pH, 9.0) for 30 min in amicrowave followed by 30 min of cooling in EDTA buffer. Sections werethen blocked in 1% horse serum for 40 minutes followed by incubationwith the 9FY monoclonal antibody at a dilution of 1:1280 for 60 min atroom temperature. Sections were incubated with the biotinylated horseanti-mouse antibody at a dilution of 1:200 (Vector Laboratories,Burlingame, Calif.) for 30 min followed by treatment with the ABC Kit(Vector Laboratories, Burlingame, Calif.) for 30 min. The colordetection was achieved by treatment with VIP (Vector Laboratories,Burlingame, Calif.) for 5 min. Sections were then counterstained inhematoxylin for 1 min, dehydrated, cleared and mounted. The 9FY stainingwas determined according to percent of cells positive: up to 25%(1+), >25-50% (2+), >50-75% (3+) and, >75% (4+). The intensity wasscored as mild (1+), moderate (2+) and marked (3+). A combination ofboth measurements was calculated by multiplying the percent of positivecells with the degree of intensity, which resulted in a score.

Analysis of ERG mRNA by Branched-Chain DNA (bDNA) Signal Amplification.

One 4 μm-thick section was selected from each of the 35 FFPE whole mountprostate samples. Areas identified as tumors were marked, removed byscraping and homogenized in 600 μl Tissue Homogenization Solution (THS)followed by the addition of 12 μl (50 μg/ml) proteinase K. Specimenswere incubated for 7 hrs at 65° C. The samples were centrifuged for 5minutes at room temperature to pellet any debris. Supernatants weretransferred to fresh microfuge tubes, avoiding any residual paraffin.All supernatants were analyzed immediately using the QUANTIGENE® 2.0Assay (Panomics, Fermont, Calif.). Each sample was assayed in duplicate.Forty μl (5 ng) of the homogenate were used for the amplification ofERG: 80 μl (10 ng) for TMPRSS2-ERG and 5 μl (0.625 ng) for housekeepinggenes: ACTB, B2M and RPL19. In addition, VCaP mRNA was used for positivecontrols. To capture target RNAs, sample dilutions were prepared bycombining appropriate volumes of samples in THS. A working probe set foreach target was prepared by combining 12 μl of the probe set with 40 μlof the blocking reagent (for target genes only) or 40 μl ofnuclease-free water (for 28S RNA), lysis mixture and nuclease-free. Theworking probe set was mixed and kept at room temperature. To prepare thecapture plate, 60 μl of each working probe set was transferred toassigned wells. Assay control and RNA samples were then made up to 100μl with Hybridization Working Reagent (HWR) and hybridized overnight at55° C. The plates were washed three times with wash buffer. Preamplifierwas then added followed by amplifier, labeled probe and finally thechemi-luminescence substrate with washes after incubation in eachreagent. The read-outs were measured in Modulus Luminometer (Knudsen etal., 2008).

Analysis of 35 evaluable specimens revealed a strong correlation betweenmRNA levels of TMPRSS2-ERG fusion type A transcript and ERG oncoproteinimmunohistochemistry (FIG. 15). A concordance of 82.8% between mRNA andprotein data was evident despite the expected differences in thesensitivity as well as read-outs of the two techniques. A comparativeevaluation of TMPRSS2-ERG gene fusion analysis by fluorescence in situhybridization (FISH) and ERG oncoprotein expression by IHC (using the9FY antibody) also supported these findings and revealed nodiscrepancies.

Example 8. Expression Map of ERG Oncoprotein in Multi-Focal ProstateCancer

Prevalent gene fusions involving regulatory sequences of the androgenreceptor (AR) regulated prostate associated genes (predominantly TMPRSS2and to a lesser extent SLC45A3 and NDRG1) and protein coding sequencesof nuclear transcription factors in the ETS gene family (primarily ERG),result in frequent overexpression of ERG in prostate tumors. Emergingstudies suggest oncogenic functions of ERG and ETVin prostate cancer(CaP) (reviewed in Kumar-Sinha, C. et al., 2008; Klezovitch, O. et al.,2008; Sun, C. et al., 2008; Tomlins, S. A. et al., 2008; Carver, B. S.et al., 2009; King, J. C. et al., 2009; Zong, Y. et al., 2009). Previousstudies including our report have analyzed ERG gene fusions at genomicor mRNA levels in the context of multi-focal cancer and these datashowed inter-tumoral heterogeneity within the same prostate (Clark, J.et al., 2007; Barry, M. et al., 2007; Mehra R. et al. 2007; Furusato, B.et al., 2008). Despite numerous reports of gene fusions and mRNAexpression, ERG oncoprotein in CaP still remains to be defined.

A monoclonal antibody directed against the ERG 42-66 epitope was used toanalyze ERG oncoprotein expression in the context of multi-focal CaP.The radical prostatectomy specimens of 132 patients enrolled in a Centerfor Prostate Disease Research program were obtained by pathologistswithin 30 minutes after the specimen was surgically removed. Theprostates were processed as whole-mounts as discussed above in Example 7From each patient, one whole mount cross section containing one to fourtumors (mostly two foci) was selected and tumors represented differentgrades and stages. Each tumor was separately diagnosed in theprostatectomy specimens and slices with more than one tumor focusrepresented separate tumors.

Each tumor was individually measured and graded. On average, one wholemount section (3.5×2.5 cm or 4.0×3.5 cm) is equivalent to approximately800-1400 tissue micro-array cores of 1 mm diameter. In addition to indextumors, most of these cross sections contained benign prostatic tissueof the peripheral and the transition/periurethral zone as well as theurethra, utricle, ejaculatory ducts, and seminal vesicles. A singletumor was present in 51 sections, and multiple individual tumors werepresent in 81 sections. Tumor grade, pathologic stage, margin status andclinical data are summarized in Table 1.

TABLE 1 Clinico-pathological features for 132 prostate cancer patients AVariables n % Race Caucasian 105 79.6 African American 27 20.4Pathological T stage pT2 34 25.8 pT3-4 84 63.6 pT2r1(x) ¹ 14 10.6Prostatectomy Specimen-Gleason Score ² 6 33 25.6 7 59 45.7 8 to 10 3728.7 Margin status Negative 70 59.3 Positive 48 40.7 Tumor grade ³Gleason pattern 3 (Well diff.) 160 61.3 Gleason pattern 4/5(Moderately/Poorly diff) 101 38.7 Biochemical recurrence ⁴ No 76 65.5Yes 40 34.5 B Variables n Mean ± SD Median (range) Age at surgery (year)132 61.1 ± 7.4  62.3 (40.2-75.2) Pretreatment PSA ng/ml) 132 7.7 ± 4.86.4 (1.1-31.4) Total tumor volume (cc) 132 10.4 ± 9.4  7.2 (0.03-52.8)Follow up months after 130 66.5 ± 35.6 67.6 (2.7-159.3) surgery

Table 1. Clinico-Pathological Features of Prostate Cancer Patients.

(A) Demographics and clinico-pathological features (categorical) ofprostate cancer patients. ¹Data from patients with pT2r1(x) tumors werenot used in this analysis. ²Corresponds to Gleason score of the indextumor represented in the section except for three patients (Case number83, 112 and 132). ³Histological appearance in the observed areas. ⁴Twoconsecutive PSAs≥0.2 ng/ml. (B) Demographics and clinico-pathologicalfeatures (continuous) of prostate cancer patients.

In both prostatic adenocarcinomas and in PIN, the epithelial cellsshowed nuclear staining. ERG was positive in 117 of 261 (44.8%)individual tumors (Table 2). Nuclear ERG staining is virtually absent inbenign epithelial cells with only 22 of about 200,000 individual benignglands staining ERG positive, demonstrating the remarkable specificity(>99.9%) of this ERG monoclonal antibody in detecting ERG-positivecarcinoma (Table 2). The number of benign glands represents an estimatebased on counting of the number of benign glands in three average sizesections of this cohort (average 1550 benign glands/section) multipliedby 132 sections.

TABLE 2 Frequency of ERG Oncoprotein Expression in Whole MountedProstatectomy Specimens ERG Individual Tumors Benign Glands Positive 11722 Negative 144 200,000 Specificity = 99.99%; Sensitivity = 44.83%; PPV84.17%; NPV = 99.93%

Of 132 specimens, only six specimens showed rare ERG positivenon-malignant cells. In three specimens, a single group of benign glands(average 7 glands, raging from 5 to 8 glands) each was positive for ERGin addition to carcinoma. In three additional specimens, ERG was presentin small aggregates of native glands (3 to 5 glands) with increasedcellularity and nuclear enlargement and mild atypia, changes previouslyreferred to as “low grade PIN.” Eight of the nine anterior/transitionzone tumors were negative. In all but five cases, over 85% of tumorcells showed moderate to strong nuclear staining with cytoplasmic blush.

Eighty-two of eighty-five (96.5%) evaluable specimens with ERG positivetumor foci contained ERG positive prostatic intraepithelial neoplasia(PIN) lesions, and all of the ERG positive PIN foci were co-located withERG positive tumors. PIN is a premalignant proliferation arising withinthe prostate.

Eighty-one sections contained multiple tumors; in 15 of these all tumorswere positive; in 31 all tumor foci were negative; and in 35 some tumorswere diffusely positive and others completely negative. Thus, in amulti-focal tumor context, 50 of 81 sections (61.7%) had one or more ERGpositive tumors. In the 51 sections containing only one tumor, 36(70.6%) were ERG positive, and two of these contained clones ofcompletely ERG negative tumor cells embedded in the positive areas. Aweak non-discriminatory cytoplasmic staining was observed in allepithelial cell types (prostatic and non-prostatic) which was consistentwith the cell line data.

The 9FY antibody consistently detected ERG in the nuclei of allendothelial cells (lympho/vascular), which served as intrinsic positivecontrol for the ERG-IHC assay. ERG expression in endothelial cells hasalso been noted previously in other contexts, however its significanceremains to be defined (Baltzinger M. et al., 1999; Birdsey, G. M. etal., 2008); Ellet, F. et al., 2009). Endothelial cells can be easilyidentified by ERG positive nucleus without or with very littlediscernible cytoplasm in contrast to carcinoma in which most of thetumor cells have ERG positive nuclei and easily identifiable cytoplasm.In ERG negative poorly differentiated/Gleason pattern 4 or 5 carcinomas,positive nuclei of endothelial cell generally have a linear, narrowdistribution.

Tumor cells with amphophilic cytoplasm were more strongly positive thanthose with pale or foamy cytoplasm. Three of the four mucin producingtumors were positive for ERG. Only two of the five tumors with a ductalcomponent were positive for ERG. One tumor with vacuolated/signetring-like appearance was positive for ERG. The focus withlymphoepithelioma-like features was negative.

In all seven patients with lymph node metastases at the time ofprostatectomy, the ERG expression mirrored the expression status of theindex tumor. Four ERG positive primary tumors had ERG positivemetastases, and conversely three ERG negative primary tumors had ERGnegative metastases. By FISH assay, ERG positive primary tumors and thecorresponding metastases showed identical fusion patterns.

Basal cells, urothelial cells of the prostatic urethra and periurethralprostatic ducts were non-reactive. Ejaculatory ducts, seminal vesicles,nerve bundles, fibromuscular stroma, variants of glandular hyperplasiaincluding microacinar hyperplasia (synonyms: adenosis, atypicaladenomatous hyperplasia), sclerosing adenosis, and basal cellhyperplasia were all negative for ERG. Different patterns of atrophyincluding proliferative inflammatory atrophy and evolving or partialatrophy were also negative for ERG.

Association of the ERG oncoprotein status was evaluated with variousclinico-pathological features (Table 1A and B). Although, ERG expressiondid not show correlation with most clinico-pathologic features, when allof the tumor foci in a given whole-mount section were taken intoaccount, higher Gleason sum and less differentiated tumors showedsignificant correlation with ERG positive immunostaining (Table 3).

TABLE 3 Association of ERG Oncoprotein Status with Tumor Differentiationand Gleason pattern of Individual Tumors (N = 261) ERG status NegativePositive Tumor grade (N = 144) (N = 117) P value Tumor differentiationGleason pattern 3 100 (62.5%) 60 (37.5%) 0.0027 (Well differentiated)Gleason pattern 4/5 44 (43.6%) 57 (56.4%) (Moderate/Poorlydifferentiated) Tumor Gleason sum 0.0094 6 100 (62.5%) 60 (37.5%) 7 26(41.3%) 37 (58.7%) 8-10 18 (47.4%) 20 (52.6%)

Statistical Analysis.

Sensitivity and specificity ofERG oncoprotein expression were analyzedfor distinguishing all tumor foci from benign glands in 132 wholemounted prostates (227 tumor foci and over 200,000 benign glands).Chi-square test or Fisher exact test were used to examine theassociation between index tumor ERG oncoprotein status with categoricalclinico-pathological features, such as race, pathological T stage,prostatectomy Gleason score and margin status. Chi square test was alsoused to test the association of ERG oncoprotein status with tumordifferentiation for individual tumors. P value of 0.05 was adopted asstatistically significant. The SAS version 9.2 was used for all dataanalysis.

Since the gene fusion events in CaP commonly involve regulatorysequences of androgen regulated prostate associated genes, e.g. TMPRSS2,SLC45A3 or NDRG1 along with protein coding sequences of the nucleartranscription factors in the ETS gene family (ERG. ETV1, ETV4-6 andELK4), the resultant protein products are ETS related oncogenictranscriptions factors with ERG being the most common (Kumar-Sinha, C.et al.; 2008) A monoclonal antibody directed against the ERG 42-66epitope exhibits a high degree of specificity and sensitivity inrecognizing ERG oncoprotein. Positive nuclear staining for the ERGoncoprotein is highly specific (>99.9%) in identifying tumor cells in65% of patients. Nuclear ERG staining is virtually absent in benignepithelial cells.

Overall 44.8% of all 261 individual tumors were ERG positive in thiscohort, whereas 70.6% of 51 specimens with a single tumor were ERGpositive and 62% of 81 specimens with more than one tumor were ERGpositive. Overall frequencies of ERG expression in CaP specimens notedhere are similar to the reported rate of gene fusions involving ERGlocus (Kumar-Sinha, C. et al., 2008; Clark, J. et al., 2007). Further,this study points to the potential contribution of sample bias inassessing frequency of ERG alterations in CaP.

In general, tumors are either homogeneously positive or negative for ERGexpression. This study highlights the association (96.5%) of ERGpositive PINs with ERG positive tumors. While other studies(Kumar-Sinha, C. et al., 2008; Cerveira et al., 2006; Perner et al.,2007) have shown lower frequency of ERG fusion positive PIN (15-20%),this study of whole-mount prostate sections allows more comprehensiveevaluation of PIN and tumors in the context of ERG oncoproteinexpression.

The rare ERG positive benign glands and the rare atypical native glands,referred to as low grade PIN, may harbor sub-morphological molecularalterations, particularly in view of their topographical relationship toPIN and/or carcinoma. This finding is in agreement with previous studiesreporting the presence of TMPRSS2-ERG fusion transcripts in rareinstances of benign prostatic glands (Clark, J. et al., 2007; Furusato,B. et al., 2008). The confirmation of TMPRSS2-ERG fusions in these fociis challenging due to their small size. When considering the highconcordance rate between ERG oncoprotein expression and TMPRSS2-ERG genefusion transcript status, one could employ the ERG IHC as an excellentsurrogate marker for gene fusions leading to ERG overexpression. Thus,in addition to complementing genomic and mRNA based assays, ERGoncoprotein detection using the antibodies described herein that bind tothe 42-66 epitope provides a significant advance in assessing ERGalterations in CaP. For example, translational products resulting fromgenomic fusion events of ERG protein coding sequence and regulatorysequence of any 5′ fusion partners (TMPRSS2. SLC45A3 and NDRG1)(Petrovics, G. et al., 2005; Han, B. et al., 2008; Pflueger, D. et al.,2009) can be detected by ERG-MAb. On the practical side, evaluation ofERG protein by IHC will be more rapid and informative for morphologicassessment of ERG oncogenic activation in a “front-end” pathologysetting.

Among the currently used diagnostic markers, alpha-methylacyl-CoAracemase (AMACR) detects approximately 80% of prostatic carcinomas and avariety of other carcinomas (Hameed, O. et al., 2005). However, thespecificity of AMACR is lower than that of the ERG, because 25-30% ofbenign prostatic glands may stain for AMACR. Thus, inclusion of anantibody that binds to the ERG 42-66 epitope in a diagnostic IHC panelcan increase the specificity for tumor detection. The strong positivereaction of the 9FY antibody in endothelial cells highlights many morecapillaries in the prostate than were previously appreciated usingconventional endothelial cell markers (CD 31, CD 34, and Factor VIIIrelated antigens). This feature of ERG expression could potentiallycomplicate the interpretation of the ERG IHC staining. For example,capillaries in intimate contact with glands may suggest basal cellstaining, or dilated capillaries with reactive endothelium may mimicsmall tumor glands or atrophy. However, this potential complication canbe overcome through experience recognizing ERG positive vascularpatterns.

While prognostic features of ERG alterations in CaP remain to be betterunderstood, both positive and negative associations have been reported(Kumar-Sinha, C. et al., 2008; Clark, J. et al., 2007). In thisevaluation of ERG oncoprotein, when all of the tumor foci in a givenwhole-mount section were taken into account, higher Gleason sum and lessdifferentiated tumors showed correlation with ERG immunostaining (Table3). However, there was no significant correlation with progression (datanot shown). Considering the ERG expression in the multi-focal tumorcontext, further independent evaluations in larger and better definedcohorts are warranted.

In summary, among the currently known CaP protein biomarkers thedetection of the homogeneous, strong and highly specific ERG oncoproteinoffers unprecedented opportunities in CaP diagnostic setting. Thesefindings substantiate the role of ERG activation in clonal selection andexpansion of ERG positive tumor cells during the transition frompre-invasive to invasive CaP in two thirds of patients. Finally, with abetter understanding of ERG functions in prostate tumor biology,ERG-monoclonal antibody based stratification of prostate tumors infuture may be used in the context of imaging, targeted therapy ormonitoring efficacy of androgen ablation therapy.

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While thisinvention has been particularly shown and described with references topreferred embodiments thereof, it will be understood by those skilled inthe art that various changes in form and details may be made thereinwithout departing from the scope of the invention encompassed by theappended claims.

REFERENCES

The following references are cited in the application and providegeneral information on the field of the invention and provide assays andother details discussed in the application. The following references areincorporated herein by reference in their entirety.

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1.-9. (canceled)
 10. A composition comprising a polypeptide having nomore than 60 amino acid residues, wherein the polypeptide includes aminoacid residues 42-66 of SEQ ID NO: 1, and wherein the composition furthercomprises an adjuvant.
 11. The composition of claim 10, wherein thepolypeptide has no more than 40 amino acid residues.
 12. The compositionof claim 10, wherein the polypeptide has no more than 25 amino acidresidues. 13-14. (canceled)
 15. The composition of claim 10, furthercomprising a pharmaceutically acceptable excipient.
 16. A method ofgenerating a monoclonal antibody that binds to the ERG 42-66 epitope,comprising administering the composition of claim 10 to a non-humanmammal, isolating B cells from the non-human mammal, immortalizing the Bcells to create a cell line capable of producing a monoclonal antibody,and selecting the monoclonal antibody that binds to the ERG 42-66epitope.
 17. (canceled)
 18. A composition comprising a polypeptidehaving no more than 60 amino acid residues, wherein the polypeptideincludes amino acid residues 42-66 of SEQ ID NO: 1, and wherein thecomposition further comprises a hapten coupled to the polypeptide. 19.The composition of claim 18, wherein the polypeptide has no more than 40amino acid residues.
 20. The composition of claim 18, wherein thepolypeptide has no more than 25 amino acid residues.
 21. The compositionof claim 18, further comprising a pharmaceutically acceptable excipient.22. A method of generating a monoclonal antibody that binds to the ERG42-66 epitope, comprising administering the composition of claim 18 to anon-human mammal, isolating B cells from the non-human mammal,immortalizing the B cells to create a cell line capable of producing amonoclonal antibody, and selecting the monoclonal antibody that binds tothe ERG 42-66 epitope.